Novel antibodies specifically binding to zika virus epitopes and uses thereof

ABSTRACT

The invention relates to antibodies, and antigen binding fragments thereof, that potently neutralize infection of ZIKV. The invention also relates to antigenic sites to which the antibodies and antigen binding fragments bind, as well as to nucleic acids that encode and immortalized B cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in screening methods as well as in the diagnosis, prophylaxis and treatment of ZIKV infection.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 470082_407 D1_SEQUENCE-LISTING.txt. The textfile is 175 kb, was created on May 26, 2021, and is being submittedelectronically via EFS-Web.

The present invention relates to antibodies, and antigen bindingfragments thereof, that bind specifically to Zika virus (ZIKV) epitopes.Such antibodies (i) potently neutralize infection of Zika virus (ZIKV)or (ii) are directed against NS1 ZIKV and can be used as diagnostics.The invention also relates to antigenic sites to which the antibodiesand antigen binding fragments bind to, as well as to nucleic acids thatencode the antibodies and immortalized B cells that produce suchantibodies and antibody fragments. In addition, the invention relates tothe use of the antibodies and antibody fragments of the invention inscreening methods as well as in the diagnosis, prevention and treatmentof ZIKV infection.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a public healthemergency. ZIKV was first isolated from macaques in 1947 in the Zikaforest in Uganda (G. W. A. Dick, S. F. Kitchen, A. J. Haddow, Zikavirus. I. Isolations and serological specificity. Trans. R. Soc. Trop.Med. Hyg. 46, 509-520 (1952)) and the first human infection was reportedin Nigeria in 1954 F. N. Macnamara, Zika virus: a report on three casesof human infection during an epidemic of jaundice in Nigeria. Trans. R.Soc. Trop. Med. Hyg. 48, 139-145 (1954)). Since then, ZIKV infectionswere sporadically reported in Africa and southeast Asia (D. Musso, VanMai Cao-Lormeau, D. J. Gubler, Zika virus: following the path of dengueand chikungunya? The Lancet. 386, 243-244 (2015)), but epidemics werereported in Micronesia in 2007 (M. R. Duffy et al., Zika virus outbreakon Yap Island, Federated States of Micronesia. N Engl J Med. 360,2536-2543 (2009)) and in French Polynesia in 2013-14, with the virussubsequently spreading to other countries in the Oceanian continent(V.-M. Cao-Lormeau, D. Musso, Emerging arboviruses in the Pacific.Lancet. 384, 1571-1572 (2014); D. Musso, E. J. Nilles, V.-M.Cao-Lormeau, Rapid spread of emerging Zika virus in the Pacific area.Clin. Microbiol. Infect. 20, 0595-6 (2014)). After its introduction intoBrazil in 2015, ZIKV has spread rapidly and in February 2016 the WorldHealth Organization (WHO) declared it a Public Health Emergency ofInternational Concern (L. R. Baden, L. R. Petersen, D. J. Jamieson, A.M. Powers, M. A. Honein, Zika Virus. N. Engl. J. Med. 374, 1552-1563(2016); A. S. Fauci, D. M. Morens, Zika Virus in the Americas—YetAnother Arbovirus Threat. N Engl J Med, 160113142101009 (2016); D. L.Heymann et al., Zika virus and microcephaly: why is this situation aPHEIC? Lancet. 387, 719-721 (2016)). The main route of ZIKV infection isthrough bites by Aedes mosquitos, but the virus may also be sexually (D.Musso et al., Potential sexual transmission of Zika virus. Emerg InfectDis. 21, 359-361 (2015)) and vertically transmitted (J. Mlakar et al.,Zika Virus Associated with Microcephaly. N Engl J Med. 374, 951-958(2016)). While most of the ZIKV infections are asymptomatic or causeonly mild symptoms, there is evidence that ZIKV infection can lead toneurological complications, such as Guillain-Barre Syndrome in adults(V.-M. Cao-Lormeau et al., Guillain-Barré Syndrome outbreak associatedwith Zika virus infection in French Polynesia: a case-control study.Lancet. 0 (2016), doi:10.1016/S0140-6736(16)00562-6) and congenitalbirth defects including microcephaly in the developing fetus G. Calvet,R. S. Aguiar, A. Melo, S. A. Sampaio, Detection and sequencing of Zikavirus from amniotic fluid of fetuses with microcephaly in Brazil: a casestudy. Lancet Infect Dis (2016), doi:10.1016/S1473-3099(16)00095-5; J.Mlakar et al., Zika Virus Associated with Microcephaly. N Engl J Med.374, 951-958 (2016); E. J. Rubin, M. F. Greene, L. R. Baden, Zika Virusand Microcephaly. N Engl J Med (2016), doi:10.1056/NEJMe1601862), likelythrough its ability to infect human neural progenitor cells H. Tang etal., Zika Virus Infects Human Cortical Neural Progenitors and AttenuatesTheir Growth. Stem Cell, 1-5 (2016)).

ZIKV belongs to the genus flavivirus, which also includes the West Nilevirus, dengue virus, tick-borne encephalitis virus, yellow fever virus,and several other viruses which may cause encephalitis. Flaviviruses areenveloped, with icosahedral and spherical geometries. The diameter isaround 50 nm. Genomes are linear positive-sense RNA and non-segmented,around 10-11 kb in length. The genome of flaviviruses encodes 3structural proteins (Capsid, prM, and Envelope) and 8 non-structuralproteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NSSB).

While flavivirus envelope (E) proteins mediate fusion and are the maintarget of neutralizing antibodies, the non-structural protein 1 (NS1) issecreted by infected cells and is involved in immune evasion andpathogenesis (D. A. Muller, P. R. Young, The flavivirus NS1 protein:molecular and structural biology, immunology, role in pathogenesis andapplication as a diagnostic biomarker. Antiviral Res. 98, 192-208(2013)). Two recent structural studies showed a high level of structuralsimilarity between the E protein of ZIKV and that of other flaviviruses,such as dengue virus (DENV), yellow fever virus (YFV) and West Nilevirus (WNV) but also revealed unique features that may be related to theZIKV neurotropism (L. Dai et al., Structures of the Zika Virus EnvelopeProtein and Its Complex with a Flavivirus Broadly Protective Antibody.Cell Host Microbe (2016), doi:10.1016/j.chom.2016.04.013; D. Sirohi etal., The 3.8 Å resolution cryo-EM structure of Zika virus. Science,aaf5316 (2016)). Similarly, the structural analysis of ZIKV NS1 revealedconserved features with NS1 of other flaviviruses although withdifferent electrostatic characteristics (J. Kim et al., Zika virus NS1structure reveals diversity of electrostatic surfaces amongflaviviruses, 1-6 (2016)).

A phenomenon that is characteristic of certain flaviviruses is thedisease-enhancing activity of cross-reactive antibodies elicited byprevious infection by heterologous viruses. In the case of Dengue virus(DENV), for which 4 serotypes are known, there is epidemiologicalevidence that a primary infection protects from reinfection with thesame serotype, but represents a risk factor for the development ofsevere disease upon reinfection with a different serotype (S. B.Halstead, Dengue Antibody-Dependent Enhancement: Knowns and Unknowns.Microbiol Spectr. 2, 249-271 (2014)). The exacerbated disease istriggered by E and prM-specific antibodies that fail to neutralize theincoming virus but instead enhance its capture by Fc receptor-expressing(FcR⁺) cells, leading to enhanced viral replication and activation ofcross-reactive memory T cells. The resulting cytokine storm is thoughtto be the basis of the most severe form of disease known as denguehemorragic fever/dengue shock syndrome (S. B. Halstead, Neutralizationand antibody-dependent enhancement of dengue viruses. Adv Virus Res. 60,421-467 (2003); G. Screaton, J. Mongkolsapaya, S. Yacoub, C. Roberts,New insights into the immunopathology and control of dengue virusinfection. Nat Rev Immunol. 15, 745-759 (2015). The role of antibodiesin severe dengue is supported by studies showing that waning levels ofmaternal antibodies in infants represent a higher risk for developmentof severe dengue disease (S. B. Halstead, Neutralization andantibody-dependent enhancement of dengue viruses. Adv Virus Res. 60,421-467 (2003); S. B. Halstead et al., Dengue hemorrhagic fever ininfants: research opportunities ignored. Emerging Infect Dis. 8,1474-1479 (2002); T. H. Nguyen et al., Dengue hemorrhagic fever ininfants: a study of clinical and cytokine profiles. J Infect Dis. 189,221-232 (2004); A. L. Rothman, Dengue: defining protective versuspathologic immunity. J Clin Invest. 113, 946-951 (2004)).

Recently, it was shown that most antibodies that reacted to DENVenvelope protein also bound to ZIKV, but those that recognize the majorlinear fusion-loop epitope (FLE) did not neutralize ZIKV and insteadpromoted antibody-dependent enhancement (ADE) of ZIKV infection(Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-Spaeth G,Duangchinda T, Sakuntabhai A, Cao-Lormeau V M, Malasit P, Rey F A,Mongkolsapaya J, Screaton G R: Dengue virus sero-cross-reactivity drivesantibody-dependent enhancement of infection with zika virus. NatImmunol. 2016 Jun. 23. doi: 10.1038/ni.3515.

Epub ahead of print

).

Moreover, according to the WHO, the recent increase in cases ofmicrocephaly and other neurological disorders potentially associatedwith Zika virus infection has prompted an increase in demand forlaboratory testing to detect Zika virus infection. In this context, highspecificity of the antibodies is required in order to distinguish ZIKVinfection from infection of other flaviviruses. However, known anti-Zikaantibodies are typically cross-reactive for other flaviviruses and,thus, not useful to distinguish ZIKV infection from infection of otherflaviviruses.

In view of the above, it is an object of the present invention toprovide novel antibodies, which specifically bind to ZIKV epitopes. Itis also an object of the present invention to provide potentlyneutralizing anti-ZIKV antibodies. Such antibodies do preferably notcontribute to antibody-dependent enhancement (ADE) of Zika virusinfection. It is also an object of the present invention to providehighly specific anti-ZIKV antibodies useful in diagnosis and testing ofZIKV infection and diagnosis methods using such antibodies.

The object underlying the present invention is solved by the claimedsubject matter.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isnot intended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis, attenuation, ameliorationand therapy. The terms “subject” or “patient” are used interchangeablyherein to mean all mammals including humans. Examples of subjectsinclude humans, cows, dogs, cats, horses, goats, sheep, pigs, andrabbits. In one embodiment, the patient is a human.

As used herein, the terms “antigen binding fragment,” “fragment,” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibody. Examples of antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)₂, Fv or scFv.Further, the term “antibody” as used herein includes both antibodies andantigen binding fragments thereof.

As used herein, the term “antibody” encompasses various forms ofantibodies including, without being limited to, whole antibodies,antibody fragments, in particular antigen binding fragments, humanantibodies, chimeric antibodies, humanized antibodies, recombinantantibodies and genetically engineered antibodies (variant or mutantantibodies) as long as the characteristic properties according to theinvention are retained. Human antibodies and monoclonal antibodies arepreferred and especially preferred are human monoclonal antibodies, inparticular as recombinant human monoclonal antibodies.

Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).Human antibodies can also be produced in transgenic animals (e.g., mice)that are capable, upon immunization, of producing a full repertoire or aselection of human antibodies in the absence of endogenousimmunoglobulin production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge (see, e.g.,Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555;Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., etal.,

ear Immunol. 7 (1993) 3340). Human antibodies can also be produced inphage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol.Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991)581-597). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).Preferably, human monoclonal antibodies are prepared by using improvedEBV-B cell immortalization as described in Traggiai E, Becker S,Subbarao K, Kolesnikova L, Uematsu Y, Gismondo M R, Murphy B R, RappuoliR, Lanzavecchia A. (2004): An efficient method to make human monoclonalantibodies from memory B cells: potent neutralization of SARScoronavirus. Nat Med. 10(8):871-5. The term “human antibody” as usedherein also comprises such antibodies which are modified, e.g. in thevariable region, to generate the properties according to the inventionas described herein. As used herein, the term “variable region”(variable region of a light chain (V_(L)), variable region of a heavychain (V_(H))) denotes each of the pair of light and heavy chains whichis involved directly in binding the antibody to the antigen.

Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will preferably be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass,whereby IgG1 is preferred. Antibodies of the invention may have a κ or aλ light chain. Preferably, the antibody according to the presentinvention, or the antigen binding fragment thereof, is a purifiedantibody, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.

The antibodies of the invention may thus preferably be human antibodies,monoclonal antibodies, human monoclonal antibodies, recombinantantibodies or purified antibodies. The invention also provides fragmentsof the antibodies of the invention, particularly fragments that retainthe antigen-binding activity of the antibodies. Such fragments include,but are not limited to, single chain antibodies, Fab, Fab′, F(ab′)2, Fvor scFv. Although the specification, including the claims, may, in someplaces, refer explicitly to antigen binding fragment(s), antibodyfragment(s), variant(s) and/or derivative(s) of antibodies, it isunderstood that the term “antibody” or “antibody of the invention”includes all categories of antibodies, namely, antigen bindingfragment(s), antibody fragment(s), variant(s) and derivative(s) ofantibodies.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Antibodies according to the present invention may be provided inpurified form. Typically, the antibody will be present in a compositionthat is substantially free of other polypeptides e.g., where less than90% (by weight), usually less than 60% and more usually less than 50% ofthe composition is made up of other polypeptides.

Antibodies according to the present invention may be immunogenic inhuman and/or in non-human (or heterologous) hosts e.g., in mice. Forexample, the antibodies may have an idiotope that is immunogenic innon-human hosts, but not in a human host. Antibodies of the inventionfor human use include those that cannot be easily isolated from hostssuch as mice, goats, rabbits, rats, non-primate mammals, etc. and cannotgenerally be obtained by humanization or from xeno-mice.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used alone, or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

Doses are often expressed in relation to the bodyweight. Thus, a dosewhich is expressed as

g, mg, or other unit

/kg (or g, mg etc.) usually refers to

g, mg, or other unit

“per kg (or g, mg etc.) bodyweight”, even if the term “bodyweight” isnot explicitly mentioned.

The term “specifically binding” and similar reference does not encompassnon-specific sticking.

The term “vaccine” as used herein is typically understood to be aprophylactic or therapeutic material providing at least one antigen,preferably an immunogen. The antigen or immunogen may be derived fromany material that is suitable for vaccination. For example, the antigenor immunogen may be derived from a pathogen, such as from bacteria orvirus particles etc., or from a tumor or cancerous tissue. The antigenor immunogen stimulates the body's adaptive immune system to provide anadaptive immune response. In particular, an “antigen” or an “immunogen”refers typically to a substance which may be recognized by the immunesystem, preferably by the adaptive immune system, and which is capableof triggering an antigen-specific immune response, e.g. by formation ofantibodies and/or antigen-specific T cells as part of an adaptive immuneresponse. Typically, an antigen may be or may comprise a peptide orprotein which may be presented by the MHC to T-cells.

As used herein, “sequence variant” (also referred to as “variant”)refers to any alteration in a reference sequence, whereby a referencesequence is any of the sequences listed in the “Tables of Sequences andSEQ ID Numbers” (sequence listing), i.e. SEQ ID NO: 1 to SEQ ID NO: 407.Thus, the term “sequence variant” includes nucleotide sequence variantsand amino acid sequence variants. Of note, the sequence variantsreferred to herein are in particular functional sequence variants, i.e.sequence variants maintaining the biological function of, for example,the antibody. In the context of the present invention such a maintainedbiological function is preferably the neutralization of ZIKV infection,the binding of the antibody to the ZIKV E protein and/or the binding ofthe antibody to the ZIKV NS1 protein. Preferred sequence variants arethus functional sequence variants having at least 70%, at least 75%, atleast 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to a reference sequence. The phrase “functionalsequence variant thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity”, as used herein, means (i) that the sequence variant isfunctional as described herein and (ii) the higher the % sequenceidentity, the more preferred the sequence variant. In other words, thephrase “functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity”, means in particular that the functionalsequence variant has at least 70% sequence identity, preferably at least75% sequence identity, preferably at least 80% sequence identity, morepreferably at least 85% sequence identity, more preferably at least 88%sequence identity, even more preferably at least 90% sequence identity,even more preferably at least 92% sequence identity, still morepreferably at least 95% sequence identity, still more preferably atleast 96% sequence identity, particularly preferably at least 97%sequence identity, particularly preferably at least 98% sequenceidentity and most preferably at least 99% sequence identity to therespective reference sequence.

The term “sequence variant” includes in particular such variants thatcomprise mutations and/or substitutions in comparison to the referencesequence. Exemplary variants of an Fc moiety sequence include, but arenot limited to, those that have an L to A substitution at position CH24, CH2 5, or both.

Sequence identity is usually calculated with regard to the full lengthof the reference sequence (i.e. the sequence recited in theapplication). Percentage identity, as referred to herein, can bedetermined, for example, using BLAST using the default parametersspecified by the NCBI (the National Center for BiotechnologyInformation; http://www.ncbi.nlm.nih.gov/)

Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1

.

As used herein, a “nucleotide sequence variant” has an altered sequencein which one or more of the nucleotides in the reference sequence isdeleted, or substituted, or one or more nucleotides are inserted intothe sequence of the reference nucleotide sequence. Nucleotides arereferred to herein by the standard one-letter designation (A, C, G, orT). Due to the degeneracy of the genetic code, a “nucleotide sequencevariant” can either result in a change in the respective reference aminoacid sequence, i.e. in an “amino acid sequence variant” or not.Preferred sequence variants are such nucleotide sequence variants, whichdo not result in amino acid sequence variants (silent mutations), butother non-silent mutations are within the scope as well, in particularmutant nucleotide sequences, which result in an amino acid sequence,which is at least 80%, preferably at least 90%, more preferably at least95% sequence identical to the reference sequence.

An “amino acid sequence variant” has an altered sequence in which one ormore of the amino acids in the reference sequence is deleted orsubstituted, or one or more amino acids are inserted into the sequenceof the reference amino acid sequence. As a result of the alterations,the amino acid sequence variant has an amino acid sequence which is atleast 80% identical to the reference sequence, preferably, at least 90%identical, more preferably at least 95% identical, most preferably atleast 99% identical to the reference sequence. Variant sequences whichare at least 90% identical have no more than 10 alterations, i.e. anycombination of deletions, insertions or substitutions, per 100 aminoacids of the reference sequence.

While it is possible to have non-conservative amino acid substitutions,it is preferred that the substitutions be conservative amino acidsubstitutions, in which the substituted amino acid has similarstructural or chemical properties with the corresponding amino acid inthe reference sequence. By way of example, conservative amino acidsubstitutions involve substitution of one aliphatic or hydrophobic aminoacids, e.g. alanine, valine, leucine and isoleucine, with another;substitution of one hydoxyl-containing amino acid, e.g. serine andthreonine, with another; substitution of one acidic residue, e.g.glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g. asparagine and glutamine, with another;replacement of one aromatic residue, e.g. phenylalanine and tyrosine,with another; replacement of one basic residue, e.g. lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includethe fusion to the N- or C-terminus of an amino acid sequence to areporter molecule or an enzyme.

Importantly, the alterations in the sequence variants do not abolish thefunctionality of the respective reference sequence, in the present case,e.g., the functionality of a sequence of an antibody, or antigen bindingfragment thereof, to bind to the same epitope and/or to sufficientlyneutralize infection of ZIKV. Guidance in determining which nucleotidesand amino acid residues, respectively, may be substituted, inserted ordeleted without abolishing such functionality are found by usingcomputer programs well known in the art.

As used herein, a nucleic acid sequence or an amino acid sequence“derived from” a designated nucleic acid, peptide, polypeptide orprotein refers to the origin of the nucleic acid, peptide, polypeptideor protein. Preferably, the nucleic acid sequence or amino acid sequencewhich is derived from a particular sequence has an amino acid sequencethat is essentially identical to that sequence or a portion thereof,from which it is derived, whereby “essentially identical” includessequence variants as defined above. Preferably, the nucleic acidsequence or amino acid sequence which is derived from a particularpeptide or protein, is derived from the corresponding domain in theparticular peptide or protein. Thereby, “corresponding” refers inparticular to the same functionality. For example, an “extracellulardomain” corresponds to another “extracellular domain” (of anotherprotein), or a “transmembrane domain” corresponds to another“transmembrane domain” (of another protein). “Corresponding” parts ofpeptides, proteins and nucleic acids are thus easily identifiable to oneof ordinary skill in the art. Likewise, sequences “derived from” othersequence are usually easily identifiable to one of ordinary skill in theart as having its origin in the sequence.

Preferably, a nucleic acid sequence or an amino acid sequence derivedfrom another nucleic acid, peptide, polypeptide or protein may beidentical to the starting nucleic acid, peptide, polypeptide or protein(from which it is derived). However, a nucleic acid sequence or an aminoacid sequence derived from another nucleic acid, peptide, polypeptide orprotein may also have one or more mutations relative to the startingnucleic acid, peptide, polypeptide or protein (from which it isderived), in particular a nucleic acid sequence or an amino acidsequence derived from another nucleic acid, peptide, polypeptide orprotein may be a functional sequence variant as described above of thestarting nucleic acid, peptide, polypeptide or protein (from which it isderived). For example, in a peptide/protein one or more amino acidresidues may be substituted with other amino acid residues or one ormore amino acid residue insertions or deletions may occur.

As used herein, the term “mutation” relates to a change in the nucleicacid sequence and/or in the amino acid sequence in comparison to areference sequence, e.g. a corresponding genomic sequence.

A mutation, e.g. in comparison to a genomic sequence, may be, forexample, a (naturally occurring) somatic mutation, a spontaneousmutation, an induced mutation, e.g. induced by enzymes, chemicals orradiation, or a mutation obtained by site-directed mutagenesis(molecular biology methods for making specific and intentional changesin the nucleic acid sequence and/or in the amino acid sequence). Thus,the terms “mutation” or “mutating” shall be understood to also includephysically making a mutation, e.g. in a nucleic acid sequence or in anamino acid sequence. A mutation includes substitution, deletion andinsertion of one or more nucleotides or amino acids as well as inversionof several successive nucleotides or amino acids. To achieve a mutationin an amino acid sequence, preferably a mutation may be introduced intothe nucleotide sequence encoding said amino acid sequence in order toexpress a (recombinant) mutated polypeptide. A mutation may be achievede.g., by altering, e.g., by site-directed mutagenesis, a codon of anucleic acid molecule encoding one amino acid to result in a codonencoding a different amino acid, or by synthesizing a sequence variant,e.g., by knowing the nucleotide sequence of a nucleic acid moleculeencoding a polypeptide and by designing the synthesis of a nucleic acidmolecule comprising a nucleotide sequence encoding a variant of thepolypeptide without the need for mutating one or more nucleotides of anucleic acid molecule.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols and reagents described herein as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

Antibodies Potently Neutralizing Zika Virus Infection

The present invention is based, amongst other findings, on the discoveryand isolation of antibodies that bind specifically to Zika virusepitopes. Such antibodies are either (i) highly potent in neutralizingZika virus, if directed to an antigenic site of Zika virus envelope (E)protein or to a ZIKV quaternary epitope or (ii) useful in diagnosis ofZika virus infection, if directed to Zika virus NS1 protein. Suchantibodies are desirable, as only small quantities of the antibodies arerequired in order to neutralize Zika virus. In particular, there iscurrently no prevention/treatment available for Zika virus infection.The antibodies according to the present invention are highly effectivein preventing as well as treating or attenuating Zika virus infection.Moreover, due to the specificity of the antibodies for Zika virus, theydo not elicit ADE, but rather block ADE. In diagnosis, Zika-specificantibodies provide an important tool for distinguishing Zika virusinfection from infection with other flaviviruses, such as Dengue virus.

In a first aspect the present invention provides an isolated antibody,or an antigen binding fragment thereof, that specifically binds to aZika virus epitope and neutralizes Zika virus infection.

In other words, the antibody, or the antigen binding fragment thereof,according to the present invention, reduces viral infectivity of Zikavirus.

To study and quantitate virus infectivity (or “neutralization”) in thelaboratory the person skilled in the art knows various standard“neutralization assays”. For a neutralization assay animal viruses aretypically propagated in cells and/or cell lines. In the context of thepresent invention a neutralization assay is preferred, wherein culturedcells are incubated with a fixed amount of Zika virus (ZIKV) in thepresence (or absence) of the antibody to be tested. As a readout forexample flow cytometry may be used. Alternatively, also other readoutsare conceivable, such as determining the amount of ZIKV non-structuralproteins (such as ZIKV NS1) secreted into culture supernatant. Forexample, a ZIKV nonstructural protein 1 (NS1) antigen captureenzyme-linked immunosorbent assay (ELISA)-based tissue cultureinfectious dose-50 (TCID50) test (TCID50-ELISA) may be used as analternative to the standard plaque assay for titrating Zika virus—in asimilar manner as described for dengue virus (DENV) by Li J, Hu D-M,Ding X-X, Chen Y, Pan Y-X, Qiu L-W, Che X-Y: Enzyme-linked immunosorbentassay-format tissue culture infectious dose-50 test for titrating denguevirus. PLoS ONE 2011, 6:e22553. In such an assay for example the ZIKVNS1-binding antibodies as described in the present application may beadvantageously used.

In a preferred embodiment of a ZIKV neutralization assay, culturedcells, for example Vero cells, are incubated with a fixed amount of ZIKVin the presence or absence of the antibody to be tested, for example forabout four days. After incubation, cells may be washed and furthercultivated. To measure virus infectivity, flow cytometry may be used. Tothis end, cells may be fixed, e.g. with 2% formaldehyde, permeabilizes,e.g. in PBS (phosphate buffered saline) 1% FCS (fetal calf serum) 0.5%saponin, and stained, e.g. with mouse antibody 4G2. Cells may then beincubated with a goat anti-mouse IgG conjugated to a dye, such as AlexaFluor488 and analyzed by flow cytometry.

Alternatively, viable cells may be detected by flow cytometry using forexample the WST-1 reagent (Roche). A preferred ZIKV strain to be used insuch a neutralization assay is ZIKV H/PF/2013.

The antibody and antigen binding fragment of the invention have highneutralizing potency. The concentration of the antibody required for 50%neutralization of Zika virus (IC₅₀) as compared to no-antibody controls,is, for example, up to about 3 μg/ml or up to about 1 μg/ml. Preferably,the concentration of the antibody of the invention required for 50%neutralization of ZIKV (IC₅₀) is up to about 500 ng/ml, more preferablythe concentration of the antibody of the invention required for 50%neutralization of ZIKV (IC₅₀) is up to about 250 ng/ml, even morepreferably the concentration of the antibody of the invention requiredfor 50% neutralization of ZIKV (IC₅₀) is up to about 150 ng/ml. Mostpreferably, the concentration of the antibody of the invention requiredfor 50% neutralization of ZIKV (IC₅₀) is about 100 ng/ml or less, e.g.about 90 ng/ml or less, about 80 ng/ml or less, about 70 ng/ml or less,about 60 ng/ml or less, about 50 ng/ml or less, about 45 ng/ml or less,about 40 ng/ml or less, about 35 ng/ml or less, about 30 ng/ml or less,about 25 ng/ml or less, about 20 ng/ml or less or, particularlypreferably, about 15 ng/ml or less. In particular, the concentration ofthe antibody of the invention required for 50% neutralization of ZIKV(IC₅₀) is preferably about 50 ng/ml or less. This means that only lowconcentrations of the antibody are required for 50% neutralization ofZIKV. The concentration of the antibody of the invention required for50% neutralization of ZIKV (IC₅₀) can be measured using standardneutralization assays as known to one of skill in the art or, inparticular, as described above.

In general, binding of an antibody may be assessed by use of a standardELISA (enzyme-linked immunosorbent assay), which is well-known to theskilled person. An exemplary standard ELISA may be performed as follows:ELISA plates may be coated (e.g., overnight at 4° C.) with a sufficientamount (e.g., 1 μg/ml) of the protein/complex/particle to which bindingof the antibody is to be tested (for example, for DENV binding asoutlined below, DENV E proteins and/or DENV VLPs are used), e.g. in PBS.Plates may then be blocked, e.g. with a 1% w/v solution of Bovine SerumAlbumin (BSA) in PBS, and incubated with the antibody to be tested (e.g.for about 1.5 hours at room temperature). After washing, antibodybinding can be revealed, e.g. using goat anti-human IgG coupled toalkaline phosphatase. Plates may then be washed, the required substrate(e.g., p-NPP) may be added and plates may be read, e.g. at 405 nm. Therelative affinities of antibody binding may be determined by measuringthe concentration of mAb (EC₅₀) required to achieve 50% maximal bindingat saturation. The EC₅₀ values may be calculated by interpolation ofbinding curves fitted with a four-parameter nonlinear regression with avariable slope.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention does essentially not bind to Denguevirus-like particles and/or to Dengue envelope protein. More preferably,the antibody, or an antigen binding fragment thereof, according to thepresent invention does essentially not bind to Dengue virus-likeparticles and/or to Dengue envelope protein of any of the four DENVserotypes DENV1, DENV2, DENV3 and DENV4. Thereby “essentially notbinding” means that for the antibody, or an antigen binding fragmentthereof, no EC₅₀-value up to 10² ng/ml, preferably up to 10³ ng/ml, morepreferably up to 5*10³ ng/ml, even more preferably up to 8*10³ ng/ml,and most preferably up to 10⁴ ng/ml can be determined in a standardELISA to Dengue virus-like particles (DENV VLP) and/or to Dengueenvelope protein (DENV E protein). In other words, the concentration ofthe antibody, or an antigen binding fragment thereof, required toachieve 50% maximal binding at saturation (EC₅₀) to Dengue virus-likeparticles (DENV VLP) and/or to Dengue envelope protein (DENV E protein)in a standard ELISA is typically more than 10² ng/ml, preferably morethan 10³ ng/ml, more preferably more than 5*10³ ng/ml, even morepreferably more than 8*10³ ng/ml, and most preferably more than 10⁴ng/ml.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention does not contribute toantibody-dependent enhancement (ADE) of Zika virus infection. Morepreferably, the antibody, or an antigen binding fragment thereof,according to the present invention blocks antibody-dependent enhancement(ADE) of Zika virus infection.

ADE may be assessed by a flow-cytometry based assay using, for examplecultured cells or cell lines, such as K562 cells. For example, theantibodies to be tested and ZIKV may be mixed for 1 hour at 37° C. andadded to 5000 K562 cells/well. After four days, cells may be fixed,permeabilized, and stained with m4G2, e.g. as described above forneutralization assays. The number of infected cells was determined byflow cytometry, as described above for neutralization assays.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention is a human antibody. It is alsopreferred that the antibody, or an antigen binding fragment thereof,according to the present invention is a monoclonal antibody, preferablya human monoclonal antibody. Furthermore, it is also preferred that theantibody, or an antigen binding fragment thereof, according to thepresent invention is a recombinant antibody.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, comprises an Fc moiety. Morepreferably, the Fc moiety is derived from human origin, e.g. from humanIgG1, IgG2, IgG3, and/or IgG4, whereby human IgG1 is particularlypreferred.

As used herein, the term “Fc moiety” refers to a sequence derived fromthe portion of an immunoglobulin heavy chain beginning in the hingeregion just upstream of the papain cleavage site (e.g., residue 216 innative IgG, taking the first residue of heavy chain constant region tobe 114) and ending at the C-terminus of the immunoglobulin heavy chain.Accordingly, an Fc moiety may be a complete Fc moiety or a portion(e.g., a domain) thereof. A complete Fc moiety comprises at least ahinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acidpositions 216-446). An additional lysine residue (K) is sometimespresent at the extreme C-terminus of the Fc moiety, but is often cleavedfrom a mature antibody. Each of the amino acid positions within an Fcmoiety have been numbered according to the art-recognized EU numberingsystem of Kabat, see e.g., by Kabat et al., in “Sequences of Proteins ofImmunological Interest”, U.S. Dept. Health and Human Services, 1983 and1987.

Preferably, in the context of the present invention an Fc moietycomprises at least one of: a hinge (e.g., upper, middle, and/or lowerhinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion,or fragment thereof. In preferred embodiments, an Fc moiety comprises atleast a hinge domain, a CH2 domain or a CH3 domain. More preferably, theFc moiety is a complete Fc moiety. The Fc moiety may also comprises oneor more amino acid insertions, deletions, or substitutions relative to anaturally-occurring Fc moiety. For example, at least one of a hingedomain, CH2 domain or CH3 domain (or portion thereof) may be deleted.For example, an Fc moiety may comprise or consist of: (i) hinge domain(or portion thereof) fused to a CH2 domain (or portion thereof), (ii) ahinge domain (or portion thereof) fused to a CH3 domain (or portionthereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain(or portion thereof), (iv) a hinge domain (or portion thereof), (v) aCH2 domain (or portion thereof), or (vi) a CH3 domain or portionthereof.

It will be understood by one of ordinary skill in the art that the Fcmoiety may be modified such that it varies in amino acid sequence fromthe complete Fc moiety of a naturally occurring immunoglobulin molecule,while retaining at least one desirable function conferred by thenaturally-occurring Fc moiety. Such functions include Fc receptor (FcR)binding, antibody half-life modulation, ADCC function, protein Abinding, protein G binding, and complement binding. The portions ofnaturally occurring Fc moieties, which are responsible and/or essentialfor such functions are well known by those skilled in the art.

For example, to activate the complement cascade C q binds to at leasttwo molecules of IgG1 or one molecule of IgM, attached to the antigenictarget (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94).Burton, D. R., described (Mol. Immunol. 22 (1985) 161-206) that theheavy chain region comprising amino acid residues 318 to 337 is involvedin complement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988)738-740), using site directed mutagenesis, reported that Glu318, Lys320and Lys322 form the binding site to C1q. The role of Glu318, Lys320 andLys 322 residues in the binding of C1q was confirmed by the ability of ashort synthetic peptide containing these residues to inhibit complementmediated lysis.

For example, FcR binding can be mediated by the interaction of the Fcmoiety (of an antibody) with Fc receptors (FcRs), which are specializedcell surface receptors on hematopoietic cells. Fc receptors belong tothe immunoglobulin superfamily, and were shown to mediate both theremoval of antibody-coated pathogens by phagocytosis of immunecomplexes, and the lysis of erythrocytes and various other cellulartargets (e.g. tumor cells) coated with the corresponding antibody, viaantibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J.G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs aredefined by their specificity for immunoglobulin classes; Fc receptorsfor IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA asFcαR and so on and neonatal Fc receptors are referred to as FcRn. Fcreceptor binding is described for example in Ravetch, J. V., and Kinet,J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al.,Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126(1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998)231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies(FcγR) triggers a wide variety of effector functions includingphagocytosis, antibody-dependent cellular cytotoxicity, and release ofinflammatory mediators, as well as immune complex clearance andregulation of antibody production. Therefore, Fc moieties providingcross-linking of receptors (FcγR) are preferred. In humans, threeclasses of FcγR have been characterized, which are: (i) FcγRI (CD64),which binds monomeric IgG with high affinity and is expressed onmacrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32),which binds complexed IgG with medium to low affinity, is widelyexpressed, in particular on leukocytes, is known to be a central playerin antibody-mediated immunity, and which can be divided into FcγRIIA,FcγRIIB and FcγRIIC, which perform different functions in the immunesystem, but bind with similar low affinity to the IgG-Fc, and theectodomains of these receptors are highly homologuous; and (iii) FcγRIII(CD16), which binds IgG with medium to low affinity and exists as twotypes: FcγRIIIA found on NK cells, macrophages, eosinophils and somemonocytes and T cells and mediating ADCC and FcγRIIIB, which is highlyexpressed on neutrophils. FcγRIIA is found on many cells involved inkilling (e.g. macrophages, monocytes, neutrophils) and seems able toactivate the killing process. FcγRIIB seems to play a role in inhibitoryprocesses and is found on B-cells, macrophages and on mast cells andeosinophils. Importantly, 75% of all FcγRIIB is found in the liver(Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endotheliumclears small immune complexes. Journal of Immunology 189: 4981-4988).FcγRIIB is abundantly expressed on Liver Sinusoidal Endothelium, calledLSEC, and in Kupffer cells in the liver and LSEC are the major site ofsmall immune complexes clearance (Ganesan, L. P. et al., 2012: FcγRIIbon liver sinusoidal endothelium clears small immune complexes. Journalof Immunology 189: 4981-4988).

Accordingly, in the present invention such antibodies, and antigenbinding fragments thereof, are preferred, which are able to bind toFcγRIIb, for example antibodies comprising an Fc moiety for binding toFcγRIIb, in particular an Fc region, such as, for example IgG-typeantibodies. Moreover, it is possible to engineer the Fc moiety toenhance FcγRIIB binding by introducing the mutations S267E and L328F asdescribed by Chu, S. Y. et al., 2008: Inhibition of B cellreceptor-mediated activation of primary human B cells by coengagement ofCD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology45, 3926-3933. Thereby, the clearance of immune complexes can beenhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE InChimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody WithEnhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit,American Thoracic Society International Conference Abstracts).Accordingly, in the context of the present invention such antibodies, orantigen binding fragments thereof, are preferred, which comprise anengineered Fc moiety with the mutations S267E and L328F, in particularas described by Chu, S. Y. et al., 2008: Inhibition of B cellreceptor-mediated activation of primary human B cells by coengagement ofCD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology45, 3926-3933.

On B-cells it seems to function to suppress further immunoglobulinproduction and isotype switching to say for example the IgE class. Onmacrophages, FcγRIIB acts to inhibit phagocytosis as mediated throughFcγRIIA. On eosinophils and mast cells the b form may help to suppressactivation of these cells through IgE binding to its separate receptor.

Regarding FcγRI binding, modification in native IgG of at least one ofE233-G236, P238, D265, N297, A327 and P329 reduces binding to FcγRI.IgG2 residues at positions 233-236, substituted into IgG1 and IgG4,reduces binding to FcγRI by 10³-fold and eliminated the human monocyteresponse to antibody-sensitized red blood cells (Armour, K. L., et al.Eur. J. Immunol. 29 (1999) 2613-2624). Regarding FcγRII binding, reducedbinding for FcγRIIA is found e.g. for IgG mutation of at least one ofE233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 andK414. Regarding FcγRIII binding, reduced binding to FcγRIIIA is founde.g. for mutation of at least one of E233-G236, P238, D265, N297, A327,P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 andD376. Mapping of the binding sites on human IgG1 for Fc receptors, theabove mentioned mutation sites and methods for measuring binding toFcγRI and FcγRIIA are described in Shields, R. L., et al., J. Biol.Chem. 276 (2001) 6591-6604.

Regarding binding to the crucial FcγRII, two regions of native IgG Fcappear to be critical for interactions of FcγRIIs and IgGs, namely (i)the lower hinge site of IgG Fc, in particular amino acid residues L, L,G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2domain of IgG Fc, in particular a loop and strands in the upper CH2domain adjacent to the lower hinge region, e.g. in a region of P331(Wines, B. D., et al., J. Immunol. 2000; 164: 5313-5318). Moreover,FcγRI appears to bind to the same site on IgG Fc, whereas FcRn andProtein A bind to a different site on IgG Fc, which appears to be at theCH2-CH3 interface (Wines, B. D., et al., J. Immunol. 2000; 164:5313-5318).

For example, the Fc moiety may comprise or consist of at least theportion of an Fc moiety that is known in the art to be required for FcRnbinding or extended half-life. Alternatively or additionally, the Fcmoiety of the antibody of the invention comprises at least the portionof known in the art to be required for Protein A binding and/or the Fcmoiety of the antibody of the invention comprises at least the portionof an Fc molecule known in the art to be required for protein G binding.Preferably, the retained function is the neutralization of Zika virusinfection, which is assumed to be mediated by FcγR binding. Accordingly,a preferred Fc moiety comprises at least the portion known in the art tobe required for FcγR binding. As outlined above, a preferred Fc moietymay thus at least comprise (i) the lower hinge site of native IgG Fc, inparticular amino acid residues L, L, G, G (234-237, EU numbering), and(ii) the adjacent region of the CH2 domain of native IgG Fc, inparticular a loop and strands in the upper CH2 domain adjacent to thelower hinge region, e.g. in a region of P331, for example a region of atleast 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upperCH2 domain of native IgG Fc around P331, e.g. between amino acids 320and 340 (EU numbering) of native IgG Fc.

Preferably, the antibody, or antigen binding fragment thereof, accordingto the present invention comprises an Fc region. As used herein, theterm “Fc region” refers to the portion of an immunoglobulin formed bytwo or more Fc moieties of antibody heavy chains. For example, the Fcregion may be monomeric or “single-chain” Fc region (i.e., a scFcregion). Single chain Fc regions are comprised of Fc moieties linkedwithin a single polypeptide chain (e.g., encoded in a single contiguousnucleic acid sequence). Exemplary scFc regions are disclosed in WO2008/143954 A2. Preferably, the Fc region is a dimeric Fc region. A“dimeric Fc region” or “dcFc” refers to the dimer formed by the Fcmoieties of two separate immunoglobulin heavy chains. The dimeric Fcregion may be a homodimer of two identical Fc moieties (e.g., an Fcregion of a naturally occurring immunoglobulin) or a heterodimer of twonon-identical Fc moieties.

The Fc moieties of the Fc region may be of the same or different classand/or subclass. For example, the Fc moieties may be derived from animmunoglobulin (e.g., a human immunoglobulin) of an IgG1, IgG2, IgG3 orIgG4 subclass. Preferably, the Fc moieties of Fc region are of the sameclass and subclass. However, the Fc region (or one or more Fc moietiesof an Fc region) may also be chimeric, whereby a chimeric Fc region maycomprise Fc moieties derived from different immunoglobulin classesand/or subclasses. For example, at least two of the Fc moieties of adimeric or single-chain Fc region may be from different immunoglobulinclasses and/or subclasses. Additionally or alternatively, the chimericFc regions may comprise one or more chimeric Fc moieties. For example,the chimeric Fc region or moiety may comprise one or more portionsderived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2,or IgG3 subclass) while the remainder of the Fc region or moiety is of adifferent subclass. For example, an Fc region or moiety of an Fcpolypeptide may comprise a CH2 and/or CH3 domain derived from animmunoglobulin of a first subclass (e.g., an IgG1, IgG2 or IgG4subclass) and a hinge region from an immunoglobulin of a second subclass(e.g., an IgG3 subclass). For example, the Fc region or moiety maycomprise a hinge and/or CH2 domain derived from an immunoglobulin of afirst subclass (e.g., an IgG4 subclass) and a CH3 domain from animmunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3subclass). For example, the chimeric Fc region may comprise an Fc moiety(e.g., a complete Fc moiety) from an immunoglobulin for a first subclass(e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of asecond subclass (e.g., an IgG1, IgG2 or IgG3 subclass). For example, theFc region or moiety may comprise a CH2 domain from an IgG4immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. Forexample, the Fc region or moiety may comprise a CH1 domain and a CH2domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. Forexample, the Fc region or moiety may comprise a portion of a CH2 domainfrom a particular subclass of antibody, e.g., EU positions 292-340 of aCH2 domain. For example, an Fc region or moiety may comprise amino acidsa positions 292-340 of CH2 derived from an IgG4 moiety and the remainderof CH2 derived from an IgG1 moiety (alternatively, 292-340 of CH2 may bederived from an IgG1 moiety and the remainder of CH2 derived from anIgG4 moiety).

Moreover, an Fc region or moiety may (additionally or alternatively) forexample comprise a chimeric hinge region. For example, the chimerichinge may be derived, e.g. in part, from an IgG1, IgG2, or IgG4 molecule(e.g., an upper and lower middle hinge sequence) and, in part, from anIgG3 molecule (e.g., an middle hinge sequence). In another example, anFc region or moiety may comprise a chimeric hinge derived, in part, froman IgG1 molecule and, in part, from an IgG4 molecule. In anotherexample, the chimeric hinge may comprise upper and lower hinge domainsfrom an IgG4 molecule and a middle hinge domain from an IgG1 molecule.Such a chimeric hinge may be made, for example, by introducing a prolinesubstitution (Ser228Pro) at EU position 228 in the middle hinge domainof an IgG4 hinge region. In another embodiment, the chimeric hinge cancomprise amino acids at EU positions 233-236 are from an IgG2 antibodyand/or the Ser228Pro mutation, wherein the remaining amino acids of thehinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequenceESKYGPPCPPCPAPPVAGP (SEQ ID NO: 408)). Further chimeric hinges, whichmay be used in the Fc moiety of the antibody according to the presentinvention are described in US 2005/0163783 A1.

In the present invention it is preferred that the Fc moiety, or the Fcregion, comprises or consists of an amino acid sequence derived from ahuman immunoglobulin sequence (e.g., from an Fc region or Fc moiety froma human IgG molecule). However, polypeptides may comprise one or moreamino acids from another mammalian species. For example, a primate Fcmoiety or a primate binding site may be included in the subjectpolypeptides. Alternatively, one or more murine amino acids may bepresent in the Fc moiety or in the Fc region.

Preferably, the antibody according to the present invention comprises,in particular in addition to an Fc moiety as described above, otherparts derived from a constant region, in particular from a constantregion of IgG, preferably from a constant region of IgG1, morepreferably from a constant region of human IgG1. More preferably, theantibody according to the present invention comprises, in particular inaddition to an Fc moiety as described above, all other parts of theconstant regions, in particular all other parts of the constant regionsof IgG, preferably all other parts of the constant regions of IgG1, morepreferably all other parts of the constant regions of human IgG1.

Particularly preferred sequences of constant regions are the amino acidsequences according to SEQ ID NOs: 145-148 (nucleic acid sequencesaccording to SEQ ID NOs: 149-152). Preferably, the amino acid sequenceof IgG1 CH1-CH2-CH3 is according to SEQ ID NO: 145 or a functionalsequence variant thereof, as described herein. Even more preferably, theamino acid sequence of IgG1 CH1-CH2-CH3 is according to SEQ ID NO: 146or a functional sequence variant thereof, as described herein, whereinthe “LALA” mutation is maintained.

As outlined above, a particularly preferred antibody according to thepresent invention comprises a (complete) Fc region derived from humanIgG1. More preferably, the antibody according to the present inventioncomprises, in particular in addition to a (complete) Fc region derivedfrom human IgG1 also all other parts of the constant regions of IgG,preferably all other parts of the constant regions of IgG1, morepreferably all other parts of the constant regions of human IgG1.

Without being bound to any theory, it is believed thatantibody-dependent enhancement (ADE) of Zika virus infection is broughtabout by the binding of the Fc moiety of the antibody, in particular,the Fc moiety of the heavy chain of an IgG molecule, to an Fc receptor,e.g., an Fcγ receptor on a host cell. It is thus preferred that theantibody according to the present invention, or an antigen bindingfragment thereof, comprises one or more mutations in the Fc moiety. Themutation(s) may be any mutation that reduces binding of the antibody toan Fc receptor (FcR), in particular reduces binding of the antibody toan Fcγ receptor (FcγR). On the other hand, it is preferred that theantibody according to the present invention comprises a (complete) Fcmoiety/Fc region, wherein the interaction/binding with FcRn is notcompromised. Accordingly, it is particularly preferred that the antibodyaccording to the present invention, or an antigen binding fragmentthereof, comprises one or more mutations in the Fc moiety, which (i)reduce(s) binding of the antibody to an Fcγ receptor, but do(es) notcompromise interaction with FcRn. One example of such a mutation is the“LALA” mutation described below.

In general, binding of the antibody to an Fc receptor may be assessed byvarious methods known to the skilled person, such as ELISA (Hessell A J,Hangartner L, Hunter M, Havenith C E G, Beurskens F J, Bakker J M,Lanigan C M S, Landucci G, Forthal D N, Parren P W H I, et al.: Fcreceptor but not complement binding is important in antibody protectionagainst HIV. Nature 2007, 449:101-104; Grevys A, Bern M, Foss S, BratlieD B, Moen A, Gunnarsen K S, Aase A, Michaelsen T E, Sandlie I, AndersenJ T: Fc Engineering of Human IgG1 for Altered Binding to the Neonatal FcReceptor Affects Fc Effector Functions. 2015, 194:5497-5508) orflow-cytometry (Perez L G, Costa M R, Todd C A, Haynes B F, Montefiori DC: Utilization of immunoglobulin G Fc receptors by humanimmunodeficiency virus type 1: a specific role for antibodies againstthe membrane-proximal external region of gp4.1. J Virol 2009,83:7397-7410; Piccoli L, Campo I, Fregni C S, Rodriguez B M F, Minola A,Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization andclearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.Nat Commun 2015, 6:1-9).

In general, the antibody according to the present invention may beglycosylated. N-linked glycans attached to the CH2 domain of a heavychain, for instance, can influence C1q and FcR binding, withaglycosylated antibodies having lower affinity for these receptors.Accordingly, the CH2 domain of the Fc moiety of the antibody accordingto the present invention may comprise one or more mutations, in which aglycosylated residue is substituted by a non-glycosylated residue. Theglycan structure can also affect activity e.g. differences incomplement-mediated cell death may be seen depending on the number ofgalactose sugars (0, 1 or 2) at the terminus of a glycan's biantennarychain. Preferably, the antibody's glycans do not lead to a humanimmunogenic response after administration.

Furthermore, the antibody according to the present invention can bemodified by introducing random amino acid mutations into particularregion of the CH2 or CH3 domain of the heavy chain in order to altertheir binding affinity for FcR and/or their serum half-life incomparison to unmodified antibodies. Examples of such modificationsinclude, but are not limited to, substitutions of at least one aminoacid from the heavy chain constant region selected from the groupconsisting of amino acid residues 250, 314, and 428.

Particularly preferably, the Fc moiety of an antibody of the inventioncomprises a substitution at positions CH2 4, CH2 5, or both. In general,the amino acid at positions 4 and 5 of CH2 of the wild-type IgG1 andIgG3 is a leucine (“L”). Preferably, the antibody according to thepresent invention comprises an amino acid at position CH2 4, CH2 5, orboth, that is not an L. More preferably, antibody according to thepresent invention comprises an alanine (“A”) at position CH2 4, or CH25, or both. Most preferably, the antibody according to the presentinvention comprises both, a CH2 L4A and a CH2 L5A substitution. Suchantibodies are referred to herein as a “LALA” variant. Interestingly,such a “LALA” mutation in the Fc moiety does not only result in a lackof contribution of the respective antibody in antibody-dependentenhancement (ADE) of Zika virus infection, but also blocksantibody-dependent enhancement (ADE) of Zika virus infection. Anexemplary amino acid sequence of IgG1 CH1-CH2-CH3 comprising the “LALA”mutation is according to SEQ ID NO: 146. Accordingly, the amino acidsequence of IgG1 CH1-CH2-CH3 is preferably according to SEQ ID NO: 146or a functional sequence variant thereof, as described herein, whereinthe “LALA” mutation is maintained.

Preferably, the antibody, or antigen binding fragment thereof, binds todomain III of Zika virus envelope protein (EDIII, also referred to as“DIII”). In other words, it is preferred that the, the antibody, orantigen binding fragment thereof, according to the present inventionbinds to an epitope of Zika virus envelope protein, which includes oneor more amino acid residues of domain III of Zika virus envelope protein(EDIII). ZIKV includes a nucleocapsid core, which comprisingsingle-stranded RNA wrapped by core proteins. The nucleocapsid core isencapsulated by a lipid bilayer membrane with “membrane proteins” and“envelope proteins”. ZIKV envelope protein (E protein) is the dominantantigen. The ectodomain of the envelope protein comprises three distinctdomains: E protein domain I (EDI), E protein domain II (EDII), and Eprotein domain III (EDIII). EDIII is highly conserved among differentZIKV strains (see FIG. 12 for an alignment of amino acid sequences ofEDIII of different ZIKV strains.

Accordingly, the antibody, or antigen binding fragment thereof, morepreferably binds to domain III of Zika virus envelope protein (EDIII)with EDIII having the following amino acid sequence (SEQ ID NO: 401):

TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDXQTLTPVGRLITANPVITEXTENSKMMLELDPPFGDSYIVIGXGXKKITHHWHRS

wherein X may be any (naturally occurring) amino acid. In other words,it is preferred that the, the antibody, or antigen binding fragmentthereof, according to the present invention binds to an epitope of Zikavirus envelope protein, which includes one or more amino acid residuesof SEQ ID NO: 401.

It is also preferred that the antibody, or antigen binding fragmentthereof, according to the present invention binds to domain III of Zikavirus envelope protein (EDIII) with EDIII having the following aminoacid sequence (SEQ ID NO: 407):

X₁GX₂X₃YSLCTAAFTFTKX₄PAEX₅X₆HGTVTVEX₇QYX₈GX₉DGPCKX₁₀PX₁₁QMAVDX₁₂QTLTPVGRLITANPVITEX₁₃TX₁₄NSKMMLELDPPFGDSYIVIGX₁₅GX₁₆X₁₇KITHHWHRSG

-   wherein X1 may be any (naturally occurring) amino acid, preferably    K, A, or E;    -   X2 may be any (naturally occurring) amino acid, preferably V, F,        or L;    -   X3 may be any (naturally occurring) amino acid, preferably S or        F;    -   X4 may be any (naturally occurring) amino acid, preferably I or        V;    -   X5 may be any (naturally occurring) amino acid, preferably T or        V;    -   X6 may be any (naturally occurring) amino acid, preferably L or        D;    -   X7 may be any (naturally occurring) amino acid, preferably V or        G;    -   X8 may be any (naturally occurring) amino acid, preferably A or        G;    -   X9 may be any (naturally occurring) amino acid except R,        preferably T or A;    -   X10 may be any (naturally occurring) amino acid, preferably V or        I;    -   X11 may be any (naturally occurring) amino acid, preferably A or        V;    -   X12 may be any (naturally occurring) amino acid, preferably M or        T;    -   X13 may be any (naturally occurring) amino acid, preferably S or        G;    -   X14 may be any (naturally occurring) amino acid, preferably E or        K;    -   X15 may be any (naturally occurring) amino acid, preferably V or        I;    -   X16 may be any (naturally occurring) amino acid, preferably E,        A, K, or D; and    -   X17 may be any (naturally occurring) amino acid, preferably E,        A, or K, more preferably K or A.

In other words, it is preferred that the, the antibody, or antigenbinding fragment thereof, according to the present invention binds to anepitope of Zika virus envelope protein, which includes one or more aminoacid residues of SEQ ID NO: 407.

For example, EDIII stretches from amino acid 309 to amino acid 403 ofZIKV E protein of the ZIKV H/PF/2013 strain (Genbank accession numberKJ776791). Accordingly, the antibody, or antigen binding fragmentthereof, most preferably binds to domain III of Zika virus envelopeprotein (EDIII) with EDIII having the following amino acid sequence (SEQID NO: 402):

TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRS.

In other words, it is preferred that the, the antibody, or antigenbinding fragment thereof, according to the present invention binds to anepitope of Zika virus envelope protein, which includes one or more aminoacid residues of SEQ ID NO: 402.

Surprisingly, the present inventors have found that antibodies bindingto domain III of Zika virus envelope protein (EDIII) show (i) increasedneutralization of ZIKV and (ii) decreased cross-reactivity with DENV (inparticular essentially no cross-reactivity with DENV) as compared toantibodies binding to domain I/II of Zika virus envelope protein(EDI/II).

More preferably, the antibody, or antigen binding fragment thereof,according to the present invention binds to an epitope of Zika virusenvelope protein, which includes one or more amino acid residues of thelateral ridge (LR) of EDIII and/or one or more amino acid residues ofthe EDI-EDIII hinge region. The EDIII lateral ridge and EDI-EDIII hingeregion are known to the skilled person and described, for example, inZhao, H., Fernandez, E., Dowd, K. A., Speer, S. D., Platt, D. J.,Gorman, M. J., Govero, J., Nelson, C. A., Pierson, T. C., Diamond, M.S., et al. (2016). Structural Basis of Zika Virus-Specific AntibodyProtection. Cell 166(4):1016-27 and in Kostyuchenko V A, Lim E X, ZhangS, Fibriansah G, Ng T S, Ooi J S, Shi J, Lok S M. Structure of thethermally stable Zika virus. Nature. 2016 May 19; 533(7603):425-8.Without being bound to any theory, it is assumed that (i) binding to theLR may inhibit fusion by trapping a fusion transitional state of thevirus and (ii) binding to the EDI-EDIII hinge and EDIII may hinder themovement of EDIII to form the trimeric post-fusion structure, therebyhalting membrane fusion.

Accordingly, it is preferred that the antibody, or antigen bindingfragment thereof, according to the present invention (is able to)inhibit(s) a post-attachment step of ZIKV. “Post-attachment” typicallyrefers to any step of ZIKV infection after attachment of ZIKV to thecell membrane (of the cell targeted by ZIKV). For example, the antibody,or antigen binding fragment thereof, according to the present inventionpreferably (is able to) prevent(s) membrane fusion. Furthermore, it isalso preferred that the antibody, or antigen binding fragment thereof,according to the present invention (is able to) cause(s) aggregation ofZIKV (particles). Most preferably, the antibody, or antigen bindingfragment thereof, according to the present invention (is able to) (i)inhibit(s) a post-attachment step of ZIKV and (ii) cause(s) aggregationof ZIKV (particles). It is also preferred that the antibody, or antigenbinding fragment thereof, binds to a quaternary epitope displayed on aZIKV infectious virion. Despite considerable neutralizing activity, suchantibodies show typically no detectable binding to recombinant ZIKV Eprotein or to ZIKV EDIII in a standard ELISA (as described above), i.e.if tested in vitro, in particular in purified form (i.e. ZIKV E protein“outside/without” a virion, a virus-like particle or the like). Thereby,“no detectable binding” typically means that no EC₅₀ up to 10000 ng/mlwas detected in a standard ELISA. In other words, if the EC₅₀ detectablein a standard ELISA is above 10000 ng/ml, it is referred to as “nodetectable binding”.

Therefore, such antibodies are also referred to herein as“neutralizing-non-E-binding” (NNB) antibodies. The quaternary epitopedisplayed on a ZIKV infectious virion is typically a conformationalepitope. For example, the quaternary epitope displayed on a ZIKVinfectious virion may be formed at the interface of two envelope proteinmonomers making up a dimer (“envelope dimer epitope”; EDE) or it may beformed across neighbouring dimers (“herring-bone epitope”).

In general, the antibody according to the present invention, or theantigen binding fragment thereof, preferably comprises (at least) threecomplementarity determining regions (CDRs) on a heavy chain and (atleast) three CDRs on a light chain. In general, complementaritydetermining regions (CDRs) are the hypervariable regions present inheavy chain variable domains and light chain variable domains.Typically, the CDRs of a heavy chain and the connected light chain of anantibody together form the antigen receptor. Usually, the three CDRs(CDR1, CDR2, and CDR3) are arranged non-consecutively in the variabledomain. Since antigen receptors are typically composed of two variabledomains (on two different polypeptide chains, i.e. heavy and lightchain), there are six CDRs for each antigen receptor (heavy chain:CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A singleantibody molecule usually has two antigen receptors and thereforecontains twelve CDRs. The CDRs on the heavy and/or light chain may beseparated by framework regions, whereby a framework region (FR) is aregion in the variable domain which is less “variable” than the CDR. Forexample, a chain (or each chain, respectively) may be composed of fourframework regions, separated by three CDR's.

The sequences of the heavy chains and light chains of exemplaryantibodies of the invention, comprising three different CDRs on theheavy chain and three different CDRs on the light chain were determined.The position of the CDR amino acids are defined according to the IMGTnumbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al.(2009) Nucleic Acids Res. 37, D1006-D1012).

Table 1 shows the SEQ ID NO's of the amino acid sequences of the heavychain CDR's (CDRH1, CDRH2, and CDRH3) and of the heavy chain variableregion (referred to as “VH”) of exemplary antibodies according to thepresent invention:

Antibody name CDRH₁ CDRH₂ CDRH₃ VH ZKA₁₉₀ 1 2 3 8 ZKA₁₈₅ 19 20 21 26ZKA₂₃₀ 37 38 39 44 ZKA₇₈ 55 56 57 62 ZKA₆₄ 73 74 75 80 ZKA₃ 237 238 239240 ZKA₄ 241 242 243 244 ZKA₅ 245 246 247 248 ZKA₆ 249 250 251 252 ZKA₇253 254 255 256 ZKA₈ 257 258 259 260 ZKA₇₆ 261 262 263 264 ZKA₁₁₇ 265266 267 268 ZKB₂₇ 269 270 271 272 ZKB₂₉ 273 274 275 276 ZKB₃₄ 277 278279 280 ZKB₃₉ 281 282 283 284 ZKB₄₆ 285 286 287 288 ZKB₅₃ 289 290 291292 ZKC₂₆ 293 294 295 296 ZKD₅ 297 298 299 300 ZKD₇ 301 302 303 304 ZKD₈305 306 307 308 ZKD₁₅ 309 310 311 312 ZKD₁₆ 313 314 315 316 ZKD₁₇ 317318 319 320 ZKD₂₀ 321 322 323 324 ZKA₁₃₄ 325 326 327 328 ZKA₂₄₆ 329 330331 332 ZKA₂₅₆ 333 334 335 336 ZKB₄₂ 337 338 339 340 ZKB₈₅ 341 342 343344 ZKB₄₇ 345 346 347 348 ZKC₆ 349 350 351 352 ZKA₁₆₀ 353 354 355 356ZKA₁₇₂ 357 358 359 360 ZKA₁₇₄ 361 362 363 364 ZKA₁₈₉ 365 366 367 368ZKA₁₉₅ 369 370 371 372 ZKA₂₁₅ 373 374 375 376 ZKA₂₁₈ 377 378 379 380ZKB₇₅ 381 382 383 384 ZKB₈₃ 385 386 387 388 ZKC₃ 389 390 391 392 ZKC₁₈393 394 395 396 ZKD₁ 397 398 399 400

Table 2 below shows the SEQ ID NO's of the amino acid sequences of thelight chain CDR's (CDRL1, CDRL2, and CDRL3) and of the light chainvariable region (referred to as “VL”) of exemplary antibodies accordingto the present invention:

Antibody name CDRL₁ CDRL₂ CDRL₂ long CDRL₃ VL ZKA₁₉₀ 4 5 6 7 9 ZKA₁₈₅ 2223 24 25 27 ZKA₂₃₀ 40 41 42 43 45 ZKA₇₈ 58 59 60 61 63 ZKA₆₄ 76 77 78 7981

It is thus preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises amino acidsequences having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to at least one of theCDR sequences, the VH sequence and/or the VL sequence shown in Table 1and/or in Table 2.

It is preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein at least one CDR, preferably the at leastone heavy chain CDRH3, comprises or consists of an amino acid sequenceaccording to any of SEQ ID NOs: 3, 75, 39, 21, 57, 239, 243, 247, 251,255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307,311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363,367, 371, 375, 379, 383, 387, 391, 395, and 399, or a functionalsequence variant thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to anyof SEQ ID NOs: 3, 21, 39, 57 and 75 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity. Morepreferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to anyof SEQ ID NOs: 3, 21, 39 and 75 or a functional sequence variant thereofhaving at least 70%, at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity. Even morepreferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 3 or according to SEQ ID NO: 75; or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity. It is alsopreferred that the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 21 or according to SEQ ID NO: 39; or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity.Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 3 or a functional sequence variant thereof having at least 70%,at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to any of SEQ ID NOs: 1, 19, 37, 55, 73, 237,    241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289,    293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341,    345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, and    397, or a functional sequence variant thereof having at least 70%,    at least 75%, at least 80%, at least 85%, at least 88%, at least    90%, at least 92%, at least 95%, at least 96%, at least 97%, at    least 98% or at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to any of SEQ ID NOs: 2, 20, 38, 56, 74, 238, 242, 246,    250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298,    302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350,    354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, and 398, or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to any of SEQ ID NOs: 3, 21, 39, 57, 75, 239,    243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291,    295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343,    347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, and    399, or a functional sequence variant thereof having at least 70%,    at least 75%, at least 80%, at least 85%, at least 88%, at least    90%, at least 92%, at least 95%, at least 96%, at least 97%, at    least 98% or at least 99% sequence identity.

Still more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to any of SEQ ID NOs: 1, 19, 37, 55 and 73 or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to any of SEQ ID NOs: 2, 20, 38, 56 and 74 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to any of SEQ ID NOs: 3, 21, 39, 57 and 75 or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to any of SEQ ID NOs: 1, 19, 37 and 73 or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to any of SEQ ID NOs: 2, 20, 38 and 74 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to any of SEQ ID NOs: 3, 21, 39 and 75 or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 1 or according to SEQ ID NO: 73; or    a functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 2 or according to SEQ ID NO: 74; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NO: 3 or according to SEQ ID NO: 75; or    a functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 19 or according to SEQ ID NO: 37;    or a functional sequence variant thereof having at least 70%, at    least 75%, at least 80%, at least 85%, at least 88%, at least 90%,    at least 92%, at least 95%, at least 96%, at least 97%, at least 98%    or at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 20 or according to SEQ ID NO: 38; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NO: 21 or according to SEQ ID NO: 39;    or a functional sequence variant thereof having at least 70%, at    least 75%, at least 80%, at least 85%, at least 88%, at least 90%,    at least 92%, at least 95%, at least 96%, at least 97%, at least 98%    or at least 99% sequence identity.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 1 or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 2 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NO: 3 or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to any of SEQ ID NOs: 4, 22, 40, 58 and 76 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to any of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 59, 60, 77 and    78 or a functional sequence variant thereof having at least 70%, at    least 75%, at least 80%, at least 85%, at least 88%, at least 90%,    at least 92%, at least 95%, at least 96%, at least 97%, at least 98%    or at least 99% sequence identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to any of SEQ ID NOs: 7, 25, 43, 61 and 79 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to any of SEQ ID NOs: 4, 22, 40 and 76 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to any of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 77 and 78 or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to any of SEQ ID NOs: 7, 25, 43 and 79 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 4 or according to SEQ ID NO: 76; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to any of SEQ ID NOs: 5, 6, 77 and 78 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 7 or according to SEQ ID NO: 79; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 22 or according to SEQ ID NO: 40; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to any of SEQ ID NOs: 23, 24, 41 and 42 or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 25 or according to SEQ ID NO: 43; or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 4 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to SEQ ID NO: 5 or 6, or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 7 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises CDRH1, CDRH2, and CDRH3amino acid sequences (i) according to SEQ ID NOs: 1-3; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (ii) according to SEQ ID NOs: 19-21; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(iii) according to SEQ ID NOs: 37-39; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (iv)according to SEQ ID NOs: 55-57; or functional sequence variants thereofhaving at least 70%, at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity; (v) according toSEQ ID NOs: 73-75; or functional sequence variants thereof having atleast 70%, at least 75%, at least 80%, at least 85%, at least 88%, atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity; (vi) according to SEQ IDNOs: 237-239; or functional sequence variants thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity; (vii) according to SEQ ID NOs:241-243; or functional sequence variants thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity; (viii) according to SEQ ID NOs: 245-247; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (ix) according to SEQ ID NOs: 249-251; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (x) according to SEQ ID NOs: 253-255; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(xi) according to SEQ ID NOs: 257-259; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xii)according to SEQ ID NOs: 261-263; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xiii)according to SEQ ID NOs: 265-267; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xiv)according to SEQ ID NOs: 269-271; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xv)according to SEQ ID NOs: 273-275; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xvi)according to SEQ ID NOs: 277-279; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xvii)according to SEQ ID NOs: 281-283; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xviii)according to SEQ ID NOs: 285-287; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xix)according to SEQ ID NOs: 289-291; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xx)according to SEQ ID NOs: 293-295; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxi)according to SEQ ID NOs: 297-299; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxii)according to SEQ ID NOs: 301-303; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxiii)according to SEQ ID NOs: 305-307; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxiv)according to SEQ ID NOs: 309-311; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxv)according to SEQ ID NOs: 313-315; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxvi)according to SEQ ID NOs: 317-319; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxvii)according to SEQ ID NOs: 321-323; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxviii)according to SEQ ID NOs: 325-327; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxix)according to SEQ ID NOs: 329-331; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxx)according to SEQ ID NOs: 333-335; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxi)according to SEQ ID NOs: 337-339; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxii)according to SEQ ID NOs: 341-343; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxiii)according to SEQ ID NOs: 345-347; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxiv)according to SEQ ID NOs: 349-351; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxv)according to SEQ ID NOs: 353-355; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxvi)according to SEQ ID NOs: 357-359; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxvii)according to SEQ ID NOs: 361-363; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxviii)according to SEQ ID NOs: 365-367; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxxix)according to SEQ ID NOs: 369-371; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xl)according to SEQ ID NOs: 373-375; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xli)according to SEQ ID NOs: 377-379; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xlii)according to SEQ ID NOs: 381-383; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xliii)according to SEQ ID NOs: 385-387; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xliv)according to SEQ ID NOs: 389-391; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xlv)according to SEQ ID NOs: 393-395; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; or (xlvi)according to SEQ ID NOs: 397-399; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

Accordingly, it is also preferred that the antibody, or the antigenbinding fragment thereof, according to the present invention comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences (i) according to SEQ ID NOs: 1-5 and 7; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (ii) according to SEQ ID NOs: 1-4 and 6-7; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (iii) according to SEQ ID NOs: 19-23 and 25; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (iv) according to SEQ ID NOs: 19-22 and 24-25; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (v) according to SEQ ID NOs: 37-41 and 43; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (vi) according to SEQ ID NOs: 37-40 and 42-43; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (vii) according to SEQ ID NOs: 55-59 and 61; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (viii) according to SEQ ID NOs: 55-58 and 60-61; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (ix) according to SEQ ID NOs: 73-77 and 79; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; or (x) according to SEQ ID NOs: 73-76 and 78-79; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises CDRH1, CDRH2, and CDRH3amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences(i) according to SEQ ID NOs: 1-5 and 7; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (ii)according to SEQ ID NOs: 1-4 and 6-7; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (iii)according to SEQ ID NOs: 19-23 and 25; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (iv)according to SEQ ID NOs: 19-22 and 24-25; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(v) according to SEQ ID NOs: 37-41 and 43; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(vi) according to SEQ ID NOs: 37-40 and 42-43; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(vii) according to SEQ ID NOs: 73-77 and 79; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;or (viii) according to SEQ ID NOs: 73-76 and 78-79; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises CDRH1, CDRH2, andCDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acidsequences (i) according to SEQ ID NOs: 1-5 and 7; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(ii) according to SEQ ID NOs: 1-4 and 6-7; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(iii) according to SEQ ID NOs: 73-77 and 79; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;or (iv) according to SEQ ID NOs: 73-76 and 78-79; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity.

It is also preferred that preferably, the antibody, or the antigenbinding fragment thereof, according to the present invention comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences (i) according to SEQ ID NOs: 19-23 and 25; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (ii) according to SEQ ID NOs: 19-22 and 24-25; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (iii) according to SEQ ID NOs: 37-41 and 43; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; or (vi) according to SEQ ID NOs: 37-40 and 42-43; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises CDRH1, CDRH2, and CDRH3amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences(i) according to SEQ ID NOs: 1-5 and 7; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; or (ii)according to SEQ ID NOs: 1-4 and 6-7; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

In addition, it is also preferred that the antibody, or the antigenbinding fragment thereof, according to the present invention comprises aheavy chain variable region (VH) and, optionally, a light chain variableregion (VL), wherein the heavy chain variable region (VH) comprises orconsists of an amino acid sequence according to any of SEQ ID NOs: 8,26, 44, 62, 80, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280,284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336,340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392,396, and 400; or a functional sequence variant thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity.

Moreover, it is also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises (i) aheavy chain variable region (VH) amino acid sequence according to SEQ IDNO: 8 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity and/or a light chain variable region (VL)amino acid sequence according to SEQ ID NO: 9 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(ii) a heavy chain variable region (VH) amino acid sequence according toSEQ ID NO: 26 or a functional sequence variant thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity and/or a light chain variableregion (VL) amino acid sequence according to SEQ ID NO: 27 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (iii) a heavy chain variable region (VH) amino acidsequence according to SEQ ID NO: 44 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity and/or a lightchain variable region (VL) amino acid sequence according to SEQ ID NO:45 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity; (iv) a heavy chain variable region (VH)amino acid sequence according to SEQ ID NO: 62 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityand/or a light chain variable region (VL) amino acid sequence accordingto SEQ ID NO: 63 or a functional sequence variant thereof having atleast 70%, at least 75%, at least 80%, at least 85%, at least 88%, atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity; or (v) a heavy chainvariable region (VH) amino acid sequence according to SEQ ID NO: 80 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 81 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises (i) a heavy chain variableregion (VH) amino acid sequence according to SEQ ID NO: 8 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 9 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (ii) a heavychain variable region (VH) amino acid sequence according to SEQ ID NO:26 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity and/or a light chain variable region (VL)amino acid sequence according to SEQ ID NO: 27 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(iii) a heavy chain variable region (VH) amino acid sequence accordingto SEQ ID NO: 44 or a functional sequence variant thereof having atleast 70%, at least 75%, at least 80%, at least 85%, at least 88%, atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity and/or a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 45 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; or (iv) a heavy chain variable region (VH) amino acidsequence according to SEQ ID NO: 80 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity and/or a lightchain variable region (VL) amino acid sequence according to SEQ ID NO:81 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises (i) a heavy chainvariable region (VH) amino acid sequence according to SEQ ID NO: 8 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 9 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; or (ii) aheavy chain variable region (VH) amino acid sequence according to SEQ IDNO: 80 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity and/or a light chain variable region (VL)amino acid sequence according to SEQ ID NO: 81 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises (i) a heavy chainvariable region (VH) amino acid sequence according to SEQ ID NO: 26 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 27 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; or (ii) aheavy chain variable region (VH) amino acid sequence according to SEQ IDNO: 44 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity and/or a light chain variable region (VL)amino acid sequence according to SEQ ID NO: 45 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain variableregion (VH) amino acid sequence according to SEQ ID NO: 8 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 9 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention is gZKA190, gZKA64, gZKA230, gZKA185or gZKA78, preferably the antibody, or the antigen binding fragmentthereof, is gZKA190, gZKA64, gZKA230 or gZKA185, more preferably theantibody, or the antigen binding fragment thereof, is gZKA190 or gZKA64,and most preferably the antibody, or the antigen binding fragmentthereof, is gZKA190.

The present inventors have isolated monoclonal antibody (mAb) accordingto the present invention, which are referred to herein as ZKA190, ZKA64,ZKA230, ZKA185 and ZKA78 (cf. Tables 1 and 2, Example 1). Based on thoseantibodies, in particular on the VH and VL genes of those antibodies,the terms “gZKA190”, “gZKA64”, “gZKA230”, “gZKA185” and “gZKA78”, asused herein, refer to the respective “generic” antibodies, or antigenbinding fragments thereof.

Namely, “gZKA190” refers to an antibody, or antigen binding fragmentthereof, having a CDRH1 amino acid sequence according to SEQ ID NO: 1, aCDRH2 amino acid sequence according to SEQ ID NO: 2, a CDRH3 amino acidsequence according to SEQ ID NO: 3, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 4, a CDRL2 amino acid sequence according to SEQID NO: 5 or 6, and a CDRL3 amino acid sequence according to SEQ ID NO:7. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 8 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO: 9.

“gZKA64” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 73, a CDRH2amino acid sequence according to SEQ ID NO: 74, a CDRH3 amino acidsequence according to SEQ ID NO: 75, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 76, a CDRL2 amino acid sequence according to SEQID NO: 77 or 78, and a CDRL3 amino acid sequence according to SEQ ID NO:79. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 80 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO:81.

“gZKA230” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 37, a CDRH2amino acid sequence according to SEQ ID NO: 38, a CDRH3 amino acidsequence according to SEQ ID NO: 39, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 40, a CDRL2 amino acid sequence according to SEQID NO: 41 or 42, and a CDRL3 amino acid sequence according to SEQ ID NO:43. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 44 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO:45.

“gZKA185” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 19, a CDRH2amino acid sequence according to SEQ ID NO: 20, a CDRH3 amino acidsequence according to SEQ ID NO: 21, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 22, a CDRL2 amino acid sequence according to SEQID NO: 23 or 24, and a CDRL3 amino acid sequence according to SEQ ID NO:25. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 26 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO:27.

“gZKA78” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 55, a CDRH2amino acid sequence according to SEQ ID NO: 56, a CDRH3 amino acidsequence according to SEQ ID NO: 57, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 58, a CDRL2 amino acid sequence according to SEQID NO: 59 or 60, and a CDRL3 amino acid sequence according to SEQ ID NO:61. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 62 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO:63.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention is for use as a medicament. In otherwords, the antibody, or an antigen binding fragment thereof, accordingto the present invention may be used in the preparation of a medicament.More preferably, the antibody, or an antigen binding fragment thereof,according to the present invention is for use in the prevention and/ortreatment of Zika virus infection. In other words, the antibody, or anantigen binding fragment thereof, according to the present invention maybe used in the preparation of a medicament or use in the preventionand/or treatment of Zika virus infection. This aspect is described inmore detail below.

Nucleic Acid Molecule

In another aspect, the invention also provides a nucleic acid moleculecomprising a polynucleotide encoding the antibody, or the antigenbinding fragment thereof, according to the present invention asdescribed above. Examples of nucleic acid molecules and/orpolynucleotides include, e.g., a recombinant polynucleotide, a vector,an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA,an siRNA, or a tRNA, or a DNA molecule such as a cDNA. Nucleic acidsequences encoding part or all of the light and heavy chains and CDRs ofthe antibodies of the present invention are preferred. Preferablyprovided herein are thus nucleic acid sequences encoding part or all ofthe light and heavy chains, in particular VH and VL sequences and CDRsof the exemplary antibodies of the invention. Tables 1 and 2 provide theSEQ ID numbers for the amino acid sequences of the CDRs and VH and VL ofexemplary antibodies according to the present invention.

Table 3 below provides the SEQ ID numbers for exemplary nucleic acidsequences encoding the CDRs and VH and VL of exemplary antibodiesaccording to the present invention. Due to the redundancy of the geneticcode, the present invention also comprises sequence variants of thesenucleic acid sequences and in particular such sequence variants, whichencode the same amino acid sequences.

A nucleic acid molecule is a molecule comprising, preferably consistingof nucleic acid components. The term nucleic acid molecule preferablyrefers to DNA or RNA molecules. In particular, it is used synonymouswith the term “polynucleotide”. Preferably, a nucleic acid molecule is apolymer comprising or consisting of nucleotide monomers which arecovalently linked to each other by phosphodiester-bonds of asugar/phosphate-backbone. The term “nucleic acid molecule” alsoencompasses modified nucleic acid molecules, such as base-modified,sugar-modified or backbone-modified etc. DNA or RNA molecules.

Table 3 shows exemplary nucleic acid sequences of the CDR's and theheavy chain variable region (VH) and the light chain variable region(VL) of five exemplary antibodies according to the present invention(“ZKA190”, “ZKA64”, “ZKA230”, “ZKA185”, “ZKA78”):

SEQ ID NO. Nucleic acid sequence ZKA190 CDRH₁ 10ggattcaccttcagtaaatatggc CDRH₂ 11 atatcatatgagggaagtaataaa CDRH₃ 12gcgaaatcggggacccaatactatgatactactggttatg agtataggggtttggaatactttggctacCDRL₁ 13 cagagtgttagtagcagttac CDRL₂ 14 gatgcatcc CDRL₂ 15ctcatctatgatgcatccagcagggcc long CDRL₃ 16 cagcagtatggtaggtcaaggtggaca VH17 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtaaatatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatcatatgagggaagtaataaatattatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggcagtgtattactgtgcgaaatcggggacccaatactatgatactactggttatgagtataggggtttggaatactttggctactgg ggccagggaaccctggtcaccgtctcctcagVL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagtagcagttacttagcctggtaccagcagaaacgtggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtaggtcaaggtggacattcggccaagggaccaaggtggaaat caaac ZKA185 CDRH₁ 28ggatatagttttaccagttactgg CDRH₂ 29 tttgatcctagtgactctcaaacc CDRH₃ 30gcgagaagatattgtagtagtagtagttgttatgtggaca at CDRL₁ 31 gcattgccaaataaatttCDRL₂ 32 gaggacaac CDRL₂ 33 gtcatctatgaggacaacaaacgaccc long CDRL₃ 34tactcaacagacagcagttctaatcccctgggagta VH 35gaagtgcagctggtgcagtccggagcagaggtgaaaaagcccggggagtctctgaggatctcctgtaagggttctggatatagttttaccagttactggatcacctgggtgcgccagatgcccgggaaaggcctggagtggatggcgaagtttgatcctagtgactctcaaaccaactacagcccgtccttccaaggccacgtcaccatctcagttgacaagtccatcagcactgcctacttgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagaagatattgtagtagtagtagttgttatgtggacaattggggccagggaaccctggtcaccatcttc tcag VL 36tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacaaacggccaggatcacctgctctggagatgcattgccaaataaatttgcttattggtaccggcagaagtcaggccaggcccctgttctggtcatctatgaggacaacaaacgaccctccgggatccctgagagattctctggctccagctcagggacaatggccaccttgactatcagtggggcccaggtggaggatgaagctgactaccactgttactcaacagacagcagttctaatcccctgggagtattcggcggagggaccaagctgac cgtcctag ZKA230 CDRH₁ 46ggtggctccatcagtagtgactac CDRH₂ 47 atctattacagtgggagcacc CDRH₃ 48gcgaggaggaggaagtatgattccctttgggggagttttg cttttgatatc CDRL₁ 49agctccaacatcggaggtaattat CDRL₂ 50 attaatgat CDRL₂ 51ctcatctgtattaatgatcaccggccc long CDRL₃ 52gcaacatgggatgacagcctgggtggccttgta VH 53caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcggagaccctgtccctcacctgcgcagtctctggtggctccatcagtagtgactactggagctggatccggcagcccccagggaagggactggagtggattgggtatatctattacagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccacttctccctgaagctgaactctgtgaccgctgcggacacggccgtgtattactgtgcgaggaggaggaagtatgattccctttgggggagttttgcttttgatatctggggccaagggacaatggtcacc gtctcttcag VL 54cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaggtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcctcatctgtattaatgatcaccggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtccgaggatgaggctgattattactgtgcaacatgggatgacagcctgggtggccttgtattcggcggagggaccaagct gaccgtcctag ZKA78 CDRH₁ 64ggcttcacttttagtaactatgca CDRH₂ 65 atcgggcgcaacggggactctatc CDRH₃ 66gtgaaagatctggccatccccgagtcctacagaattgaag ctgattat CDRL₁ 67cagtccgtgctgtaccgctctaacaacaagaattac CDRL₂ 68 tgggcttca CDRL₂ 69ctgatctattgggcttcaacccgggaa long CDRL₃ 70 cagcagtactattctagtcctcgaact VH71 gaggtgcagctggcagaatcaggcgggggactggtccagcctggcggcagcctgacactgtcttgcagtggatcaggcttcacttttagtaactatgcaatggtgtgggcaaggcaggctcctgggaagggactggagtatgtctctggcatcgggcgcaacggggactctatctactatactgatagtgtgaagggccggttcaccatcagcagagacaatagcaaatccatggtgtacctgcagatgagctccctgcgaaccgaagacacagcagtgtactattgcgtgaaagatctggccatccccgagtcctacagaattgaagctgattattggggacagggcaccctggtcatc gtgagcgccg VL 72gacatcgtgatgacacagtctccagatagtctggcagtcagtctgggggagagggccactattaactgcaagagctcccagtccgtgctgtaccgctctaacaacaagaattacctgtcttggtatcagcagaagcccggacagccccctaaactgctgatctattgggcttcaacccgggaaagcggcgtcccagacagattctcaggcagcgggtccggaacagacttcaccctgacaattagccccctgcaggcagaggacgtggctgtctactattgtcagcagtactattctagtcctcgaactttcggccaggg gaccaaggtggaaatcaaac ZKA64CDRH₁ 82 ggctacaccttcacagggtatcac CDRH₂ 83 attaaccctaattctggcgggaccCDRH₃ 84 gctcggatgagctcctctatttggggcttcgatcat CDRL₁ 85cagtctgtgctgattaac CDRL₂ 86 ggagcatcc CDRL₂ 87ctgatctatggagcatcctccagggct long CDRL₃ 88 cagcagtacaatgattggccccctatcacaVH 89 caggtgcagctggtccagagcggagcagaggtgaagaaacccggcgcctcagtgaaggtcagctgcaaagcttccggctacaccttcacagggtatcacatcgactgggtgaggcaggcaagaggacagggactggaatggatgggacggattaaccctaattctggcgggaccaactacgcccagaagtttcagggccgagtgactatgaccagagacaccagcatctccacagcttatatgcagctgtcccggctgagatctgacgatagtgccgtctactattgtgctcggatgagctcctctatttggggcttcgatcattgggggcagggaacactggtgactgtcagttcag VL 90gagatcgtgatgactcagtctccagccaccctgtcagtcagcccaggagaacgggcaaccctgtcttgcagagcctcccagtctgtgctgattaacctggcttggtaccagcagaagccaggccaggcaccccgactgctgatctatggagcatcctccagggctaccggcattcctgcacgcttcagtggatcaggaagcggaacagagtttaccctgacaatctctagtctgcagtccgaagacttcgctgtctactattgtcagcagtacaatgattggccccctatcacatttggccaggggactagactggagat caagc

Preferably, the sequence of the nucleic acid molecule according to thepresent invention comprises or consists of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 10-18, 28-36, 46-54, 64-72, and82-90; or a functional sequence variant thereof.

It is also preferred that nucleic acid sequences according to theinvention include nucleic acid sequences having at least 70%, at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identity to the nucleic acid encoding a CDR, a VH sequence and/or aVL sequence used in an (exemplary) antibody according to the presentinvention, for example to the sequences shown in Table 3.

In general, the nucleic acid molecule may be manipulated to insert,delete or alter certain nucleic acid sequences. Changes from suchmanipulation include, but are not limited to, changes to introducerestriction sites, to amend codon usage, to add or optimizetranscription and/or translation regulatory sequences, etc. It is alsopossible to change the nucleic acid to alter the encoded amino acids.For example, it may be useful to introduce one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/orinsertions into the antibody's amino acid sequence. Such point mutationscan modify effector functions, antigen-binding affinity,post-translational modifications, immunogenicity, etc., can introduceamino acids for the attachment of covalent groups (e.g., labels) or canintroduce tags (e.g., for purification purposes). Mutations can beintroduced in specific sites or can be introduced at random, followed byselection (e.g., molecular evolution). For instance, one or more nucleicacids encoding any of the CDR regions, a VH sequence and/or a VLsequence of an (exemplary) antibody of the invention can be randomly ordirectionally mutated to introduce different properties in the encodedamino acids. Such changes can be the result of an iterative processwherein initial changes are retained and new changes at other nucleotidepositions are introduced. Further, changes achieved in independent stepsmay be combined. Different properties introduced into the encoded aminoacids may include, but are not limited to, enhanced affinity.

Vector

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid moleculeaccording to the present invention. Preferably, a vector comprises anucleic acid molecule as described above.

The term “vector” refers to a nucleic acid molecule, preferably to arecombinant nucleic acid molecule, i.e. a nucleic acid molecule whichdoes not occur in nature. A vector in the context of the presentinvention is suitable for incorporating or harboring a desired nucleicacid sequence. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule. Thus,the vector may comprise a sequence corresponding, e.g., to a desiredantibody or antibody fragment thereof according to the presentinvention. An expression vector may be used for production of expressionproducts such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.For example, an expression vector may comprise sequences needed fortranscription of a sequence stretch of the vector, such as a promotersequence. A cloning vector is typically a vector that contains a cloningsite, which may be used to incorporate nucleic acid sequences into thevector. A cloning vector may be, e.g., a plasmid vector or abacteriophage vector. A transfer vector may be a vector which issuitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors. A vector in the context of thepresent invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector.

Cells

In a further aspect, the present invention also provides cell expressingthe antibody, or the antigen binding fragment thereof, according to thepresent invention; and/or comprising the vector according the presentinvention.

Examples of such cells include but are not limited to, eukaryotic cells,e.g., yeast cells, animal cells or plant cells. Preferably, the cellsare mammalian cells, more preferably a mammalian cell line. Preferredexamples include human cells, CHO cells, HEK293T cells, PER.C6 cells,NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, the cell may be transfected with a vector according tothe present invention, preferably with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably intoeukaryotic cells. In the context of the present invention, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, preferably intoeukaryotic cells, such as into mammalian cells. Such methods encompass,for example, electroporation, lipofection, e.g. based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine etc.Preferably, the introduction is non-viral.

Moreover, the cells of the present invention may be transfected stablyor transiently with the vector according to the present invention, e.g.for expressing the antibody, or the antigen binding fragment thereof,according to the present invention. Preferably, the cells are stablytransfected with the vector according to the present invention encodingthe antibody, or the antigen binding fragment thereof, according to thepresent invention. Alternatively, it is also preferred that the cellsare transiently transfected with the vector according to the presentinvention encoding the antibody, or the antigen binding fragmentthereof, according to the present invention.

Optional Additional Features of the Antibodies

Antibodies of the invention may be coupled, for example, to a drug fordelivery to a treatment site or coupled to a detectable label tofacilitate imaging of a site comprising cells of interest. Methods forcoupling antibodies to drugs and detectable labels are well known in theart, as are methods for imaging using detectable labels. Labeledantibodies may be employed in a wide variety of assays, employing a widevariety of labels. Detection of the formation of an antibody-antigencomplex between an antibody of the invention and an epitope of interestcan be facilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 1251, 1311, 35S, or 3H. Suchlabeled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. Labeled antibodies according to the presentinvention may be thus be used in such assays for example as described inU.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. Inaddition, linkers may be used between the labels and the antibodies ofthe invention, e.g., as described in U.S. Pat. No. 4,831,175. Antibodiesor, antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art, e.g., as described in U.S. Pat. No. 5,595,721. Treatment mayconsist of a combination of treatment with conjugated and non-conjugatedantibodies administered simultaneously or subsequently e.g., asdescribed in WO00/52031; WO00/52473.

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life.Examples of polymers, and methods to attach them to peptides, are shownin U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. In someembodiments the polymers may be selected from polyoxyethylated polyolsand polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH₂—CH₂)_(n)O—R, wherein Rcan be hydrogen, or a protective group such as an alkyl or alkanolgroup. Preferably, the protective group may have between 1 and 8carbons. For example, the protective group is methyl. The symbol n is apositive integer. In one embodiment n is between 1 and 1,000. In anotherembodiment n is between 2 and 500. Preferably, the PEG has an averagemolecular weight between 1,000 and 40,000, more preferably the PEG has amolecular weight between 2,000 and 20,000, even more preferably the PEGhas a molecular weight between 3,000 and 12,000. Furthermore, PEG mayhave at least one hydroxy group, for example the PEG may have a terminalhydroxy group. For example, it is the terminal hydroxy group which isactivated to react with a free amino group on the inhibitor. However, itwill be understood that the type and amount of the reactive groups maybe varied to achieve a covalently conjugated PEG/antibody of the presentinvention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment, POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. POG may have a molecular weight in the same range asPEG. Another drug delivery system that can be used for increasingcirculatory half-life is the liposome. Methods of preparing liposomedelivery systems are known to one of skill in the art. Other drugdelivery systems are known in the art and are described in, for example,referenced in Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g., where less than 90% (by weight), usuallyless than 60% and more usually less than 50% of the composition is madeup of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g., in mice. In particular, the antibodies mayhave an idiotope that is immunogenic in non-human hosts, but not in ahuman host. In particular, antibodies of the invention for human useinclude those that cannot be easily isolated from hosts such as mice,goats, rabbits, rats, non-primate mammals, etc. and cannot generally beobtained by humanization or from xeno-mice.

Production of Antibodies

Antibodies according to the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C., 1975; Kozbar et al. 1983). In one embodiment, thealternative EBV immortalization method described in WO2004/076677 isused.

A preferred method is described in WO 2004/076677. In this method Bcells producing the antibody of the invention are transformed with EBVand a polyclonal B cell activator. Additional stimulants of cellulargrowth and differentiation may optionally be added during thetransformation step to further enhance the efficiency. These stimulantsmay be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is addedduring the immortalization step to further improve the efficiency ofimmortalization, but its use is not essential. The immortalized B cellsproduced using these methods can then be cultured using methods known inthe art and antibodies isolated therefrom.

Another preferred method is described in WO 2010/046775. In this methodplasma cells are cultured in limited numbers, or as single plasma cellsin microwell culture plates. Antibodies can be isolated from the plasmacell cultures. Further, from the plasma cell cultures, RNA can beextracted and PCR can be performed using methods known in the art. TheVH and VL regions of the antibodies can be amplified by RT-PCR (reversetranscriptase PCR), sequenced and cloned into an expression vector thatis then transfected into HEK293T cells or other host cells. The cloningof nucleic acid in expression vectors, the transfection of host cells,the culture of the transfected host cells and the isolation of theproduced antibody can be done using any methods known to one of skill inthe art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments of the presentinvention. Desired DNA sequences may be synthesized completely or inpart using oligonucleotide synthesis techniques. Site-directedmutagenesis and polymerase chain reaction (PCR) techniques may be usedas appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g., mammalian, host cell expression systems may be usedfor production of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector encoding a nucleic acid of thepresent invention under conditions suitable for expression of proteinfrom DNA encoding the antibody molecule of the present invention, andisolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced by(i) expressing a nucleic acid sequence according to the invention in ahost cell, e.g. by use of a vector according to the present invention,and (ii) isolating the expressed antibody product. Additionally, themethod may include (iii) purifying the isolated antibody. Transformed Bcells and cultured plasma cells may be screened for those producingantibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, e.g., ELISA,by staining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function. The assay mayselect on the basis of simple recognition of one or more antigens, ormay select on the additional basis of a desired function e.g., to selectneutralizing antibodies rather than just antigen-binding antibodies, toselect antibodies that can change characteristics of targeted cells,such as their signaling cascades, their shape, their growth rate, theircapability of influencing other cells, their response to the influenceby other cells or by other reagents or by a change in conditions, theirdifferentiation status, etc.

Individual transformed B cell clones may then be produced from thepositive transformed B cell culture. The cloning step for separatingindividual clones from the mixture of positive cells may be carried outusing limiting dilution, micromanipulation, single cell deposition bycell sorting or another method known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned andexpressed in HEK293T cells or other known host cells using methods knownin the art.

The immortalized B cell clones or the transfected host-cells of theinvention can be used in various ways e.g., as a source of monoclonalantibodies, as a source of nucleic acid (DNA or mRNA) encoding amonoclonal antibody of interest, for research, etc.

The invention also provides a composition comprising immortalized Bmemory cells or transfected host cells that produce antibodies accordingto the present invention.

The immortalized B cell clone or the cultured plasma cells of theinvention may also be used as a source of nucleic acid for the cloningof antibody genes for subsequent recombinant expression. Expression fromrecombinant sources is more common for pharmaceutical purposes thanexpression from B cells or hybridomas e.g., for reasons of stability,reproducibility, culture ease, etc.

Thus the invention also provides a method for preparing a recombinantcell, comprising the steps of: (i) obtaining one or more nucleic acids(e.g., heavy and/or light chain mRNAs) from the B cell clone or thecultured plasma cells that encodes the antibody of interest; (ii)inserting the nucleic acid into an expression vector and (iii)transfecting the vector into a host cell in order to permit expressionof the antibody of interest in that host cell.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone or the cultured plasma cells that encodes the antibody ofinterest; and (ii) using the sequence information from step (i) toprepare nucleic acid(s) for insertion into a host cell in order topermit expression of the antibody of interest in that host cell. Thenucleic acid may, but need not, be manipulated between steps (i) and(ii) to introduce restriction sites, to change codon usage, and/or tooptimize transcription and/or translation regulatory sequences.

Furthermore, the invention also provides a method of preparing atransfected host cell, comprising the step of transfecting a host cellwith one or more nucleic acids that encode an antibody of interest,wherein the nucleic acids are nucleic acids that were derived from animmortalized B cell clone or a cultured plasma cell of the invention.Thus the procedures for first preparing the nucleic acid(s) and thenusing it to transfect a host cell can be performed at different times bydifferent people in different places (e.g., in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture technique can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsor plasma cells are well known in the art (e.g., see Chapter 4 of KubyImmunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells,human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a humanliver cell), as well as plant cells, whereby mammalian cells arepreferred. Preferred expression hosts can glycosylate the antibody ofthe invention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. In one embodiment the transfected hostcell may be able to grow in serum-free media. In a further embodimentthe transfected host cell may be able to grow in culture without thepresence of animal-derived products. The transfected host cell may alsobe cultured to give a cell line.

The invention also provides a method for preparing one or more nucleicacid molecules (e.g., heavy and light chain genes) that encode anantibody of interest, comprising the steps of (i) preparing animmortalized B cell clone or culturing plasma cells according to theinvention; (ii) obtaining from the B cell clone or the cultured plasmacells nucleic acid that encodes the antibody of interest.

Further, the invention provides a method for obtaining a nucleic acidsequence that encodes an antibody of interest, comprising the steps of:(i) preparing an immortalized B cell clone or culturing plasma cellsaccording to the invention; (ii) sequencing nucleic acid from the B cellclone or the cultured plasma cells that encodes the antibody ofinterest.

The invention further provides a method of preparing nucleic acidmolecule(s) that encode an antibody of interest, comprising the step ofobtaining the nucleic acid that was obtained from a transformed B cellclone or cultured plasma cells of the invention. Thus the procedures forfirst obtaining the B cell clone or the cultured plasma cell, and thenobtaining nucleic acid(s) from the B cell clone or the cultured plasmacells can be performed at different times by different people indifferent places (e.g., in different countries).

The invention also comprises a method for preparing an antibody (e.g.,for pharmaceutical use) according to the present invention, comprisingthe steps of: (i) obtaining and/or sequencing one or more nucleic acids(e.g., heavy and light chain genes) from the selected B cell clone orthe cultured plasma cells expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s)sequence(s) to prepare an expression vector; (iii) transfecting a hostcell that can express the antibody of interest; (iv) culturing orsub-culturing the transfected host cells under conditions where theantibody of interest is expressed; and, optionally, (v) purifying theantibody of interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing a transfected host cellpopulation, e.g. a stably transfected host cell population, underconditions where the antibody of interest is expressed and, optionally,purifying the antibody of interest, wherein said transfected host cellpopulation has been prepared by (i) providing nucleic acid(s) encoding aselected antibody of interest that is produced by a B cell clone orcultured plasma cells prepared as described above, (ii) inserting thenucleic acid(s) into an expression vector, (iii) transfecting the vectorin a host cell that can express the antibody of interest, and (iv)culturing or sub-culturing the transfected host cell comprising theinserted nucleic acids to produce the antibody of interest. Thus theprocedures for first preparing the recombinant host cell and thenculturing it to express antibody can be performed at very differenttimes by different people in different places (e.g., indifferentcountries).

Pharmaceutical Composition

The present invention also provides a pharmaceutical compositioncomprising one or more of:

-   (i) the antibody, or the antibody fragment thereof, according to the    present invention;-   (ii) the nucleic acid encoding the antibody, or antibody fragments    according to the present invention;-   (iii) the vector comprising the nucleic acid according to the    present invention; and/or-   (iv) the cell expressing the antibody according to the present    invention or comprising the vector according to the present    invention.

In other words, the present invention also provides a pharmaceuticalcomposition comprising the antibody, or the antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present inventionand/or the cell according to the present invention.

The pharmaceutical composition may preferably also contain apharmaceutically acceptable carrier, diluent and/or excipient. Althoughthe carrier or excipient may facilitate administration, it should notitself induce the production of antibodies harmful to the individualreceiving the composition. Nor should it be toxic. Suitable carriers maybe large, slowly metabolized macromolecules such as proteins,polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolicacids, polymeric amino acids, amino acid copolymers and inactive virusparticles. In general, pharmaceutically acceptable carriers in apharmaceutical composition according to the present invention may beactive components or inactive components. Preferably, thepharmaceutically acceptable carrier in a pharmaceutical compositionaccording to the present invention is not an active component in respectto Zika virus infection.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in a pharmaceutical composition mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Pharmaceutical compositions of the invention may be prepared in variousforms. For example, the compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared (e.g., a lyophilized composition, similar to Synagis™and Herceptin™, for reconstitution with sterile water containing apreservative). The composition may be prepared for topicaladministration e.g., as an ointment, cream or powder. The compositionmay be prepared for oral administration e.g., as a tablet or capsule, asa spray, or as a syrup (optionally flavored). The composition may beprepared for pulmonary administration e.g., as an inhaler, using a finepowder or a spray. The composition may be prepared as a suppository orpessary. The composition may be prepared for nasal, aural or ocularadministration e.g., as drops. The composition may be in kit form,designed such that a combined composition is reconstituted just prior toadministration to a subject. For example, a lyophilized antibody may beprovided in kit form with sterile water or a sterile buffer.

It is preferred that the active ingredient in the composition is anantibody molecule, an antibody fragment or variants and derivativesthereof, in particular the active ingredient in the composition is anantibody, an antibody fragment or variants and derivatives thereof,according to the present invention. As such, it may be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition may contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and inother embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Within the scope of the invention are compositions present in severalforms of administration; the forms include, but are not limited to,those forms suitable for parenteral administration, e.g., by injectionor infusion, for example by bolus injection or continuous infusion.Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilizing and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid. A vehicle is typically understood to be amaterial that is suitable for storing, transporting, and/oradministering a compound, such as a pharmaceutically active compound, inparticular the antibodies according to the present invention. Forexample, the vehicle may be a physiologically acceptable liquid, whichis suitable for storing, transporting, and/or administering apharmaceutically active compound, in particular the antibodies accordingto the present invention. Once formulated, the compositions of theinvention can be administered directly to the subject. In one embodimentthe compositions are adapted for administration to mammalian, e.g.,human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Preferably, the pharmaceuticalcomposition may be prepared for oral administration, e.g. as tablets,capsules and the like, for topical administration, or as injectable,e.g. as liquid solutions or suspensions, whereby it is particularlypreferred that the pharmaceutical composition is an injectable. Solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection are also be preferred, e.g. that the pharmaceuticalcomposition is in lyophilized form.

For injection, e.g. intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient willpreferably be in the form of a parenterally acceptable aqueous solutionwhich is pyrogen-free and has suitable pH, isotonicity and stability.Those of relevant skill in the art are well able to prepare suitablesolutions using, for example, isotonic vehicles such as Sodium ChlorideInjection, Ringer's Injection, Lactated Ringer's Injection.Preservatives, stabilizers, buffers, antioxidants and/or other additivesmay be included, as required. Whether it is a polypeptide, peptide, ornucleic acid molecule, other pharmaceutically useful compound accordingto the present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. For injection, thepharmaceutical composition according to the present invention may beprovided for example in a pre-filled syringe.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivetransporter cargo conjugate molecule as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive pharmaceutical composition, particularly its components asdefined above, suspended or dissolved in one or more carriers. Carriersfor topical administration include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, theinventive pharmaceutical composition can be formulated in a suitablelotion or cream. In the context of the present invention, suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

Dosage treatment may be a single dose schedule or a multiple doseschedule. In particular, the pharmaceutical composition may be providedas single-dose product. Preferably, the amount of the antibody in thepharmaceutical composition—in particular if provided as single-doseproduct—does not exceed 200 mg, more preferably does not exceed 100 mg,and even more preferably does not exceed 50 mg.

For example, the pharmaceutical composition according to the presentinvention may be administered daily, e.g. once or several times per day,e.g. once, twice, three times or four times per day, preferably once ortwice per day, more preferable once per day, for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more days, e.g.daily for 1, 2, 3, 4, 5, 6 months. Preferably, the pharmaceuticalcomposition according to the present invention may be administeredweekly, e.g. once or twice per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more weeks, e.g. weeklyfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or weekly for 2, 3,4, or 5 years. Moreover, the pharmaceutical composition according to thepresent invention may be preferably administered monthly, e.g. once permonth or, more preferably, every second month for 1, 2, 3, 4, or 5 ormore years. It is also preferred that the administration continues forthe lifetime. In addition, also one single administration only is alsoenvisaged, in particular in respect to certain indications, e.g. forprevention of Zika virus infection in case of accidental exposure, e.g.in non-immunised subjects. However, the most preferred treatmentschedule is post-exposure prophylaxis (PEP), wherein one or more singledoses are administered as soon as possible after Zika infection. Aprophylactic setting, wherein one or more single doses are administeredto prevent Zika infection (i.e. before Zika infection, in particular innon-Zika-immunised subjects) is also preferred.

In particular, it is preferred that for a single dose, e.g. a daily,weekly or monthly dose, preferably for a weekly dose, the amount of theantibody, or the antigen binding fragment thereof, in the pharmaceuticalcomposition according to the present invention, does not exceed 1 g,preferably does not exceed 500 mg, more preferably does not exceed 200mg, even more preferably does not exceed 100 mg, and particularlypreferably does not exceed 50 mg.

Pharmaceutical compositions typically include an “effective” amount ofone or more antibodies of the invention, i.e. an amount that issufficient to treat, ameliorate, attenuate or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic effect. Therapeuticeffects also include reduction or attenuation in pathogenic potency orphysical symptoms. The precise effective amount for any particularsubject will depend upon their size, weight, and health, the nature andextent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.005 to about 100 mg/kg,preferably from about 0.0075 to about 50 mg/kg, more preferably fromabout 0.01 to about 10 mg/kg, and even more preferably from about 0.02to about 5 mg/kg, of the antibody of the present invention (e.g. amountof the antibody in the pharmaceutical composition) in relation to thebodyweight (e.g., in kg) of the individual to which it is administered.

Moreover, the pharmaceutical composition according to the presentinvention may also comprise an additional active component, which may bea further antibody or a component, which is not an antibody. Theadditional active component is preferably a checkpoint inhibitor. It isalso preferred that a ZIKV neutralizing antibody, or an antigen bindingfragment thereof, as described herein is combined with a ZIKVNS1-binding antibody, or an antigen binding fragment thereof, asdescribed herein as additional active component (co-agent). Thereby, thepathogenic role of NS1 may be blocked in addition to neutralization ofZIKV. The pharmaceutical composition according to the present inventionmay comprise one or more of the additional active components, e.g. asdescribed as co-agents below in the context of a combination therapy.

The antibody, or the antigen binding fragment, according to the presentinvention can be present either in the same pharmaceutical compositionas the additional active component or, preferably, the antibody, or theantigen binding fragment, according to the present invention iscomprised by a first pharmaceutical composition and the additionalactive component is comprised by a second pharmaceutical compositiondifferent from the first pharmaceutical composition. Accordingly, ifmore than one additional active component is envisaged, each additionalactive component and the antibody, or the antigen binding fragment,according to the present invention is preferably comprised by adifferent pharmaceutical composition. Such different pharmaceuticalcompositions may be administered either combined/simultaneously or atseparate times or at separate locations (e.g. separate parts of thebody).

Preferably, antibody, or the antigen binding fragment, according to thepresent invention and the additional active component provide anadditive therapeutic effect or, preferably, a synergistic therapeuticeffect. The term “synergy” is used to describe a combined effect of twoor more active agents that is greater than the sum of the individualeffects of each respective active agent. Thus, where the combined effectof two or more agents results in “synergistic inhibition” of an activityor process, it is intended that the inhibition of the activity orprocess is greater than the sum of the inhibitory effects of eachrespective active agent. The term “synergistic therapeutic effect”refers to a therapeutic effect observed with a combination of two ormore therapies wherein the therapeutic effect (as measured by any of anumber of parameters) is greater than the sum of the individualtherapeutic effects observed with the respective individual therapies.

A pharmaceutical composition comprising the antibody according togZKA190, gZKA64, gZKA230, gZKA185, gZKA78 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier is preferred.

In one embodiment, a composition of the invention may include antibodiesof the invention, wherein the antibodies may make up at least 50% byweight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore) of the total protein in the composition. In such a composition,the antibodies are preferably in purified form.

The present invention also provides a method of preparing apharmaceutical composition comprising the steps of: (i) preparing anantibody of the invention; and (ii) admixing the purified antibody withone or more pharmaceutically-acceptable carriers.

In another embodiment, a method of preparing a pharmaceuticalcomposition comprises the step of: admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell or acultured plasma cell of the invention.

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestderived from the B cell or the cultured plasma cells to a subject, suchthat the nucleic acid can be expressed in the subject in situ to providea desired therapeutic effect. Suitable gene therapy and nucleic aciddelivery vectors are known in the art.

Pharmaceutical compositions may include an antimicrobial particularly ifpackaged in a multiple dose format. They may comprise detergent e.g., aTween (polysorbate), such as Tween 80. Detergents are generally presentat low levels e.g., less than 0.01%. Compositions may also includesodium salts (e.g., sodium chloride) to give tonicity. For example, aconcentration of 10-±2 mg/ml NaCl is typical.

Further, pharmaceutical compositions may comprise a sugar alcohol (e.g.,mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilizedor if they include material which has been reconstituted fromlyophilized material. The pH of a composition for lyophilization may beadjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 priorto lyophilization.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

Medical Treatments, Kits and Uses

Medical Treatments

In a further aspect, the present invention provides the use of theantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention in (i) prevention and/or treatment of Zika virusinfection; or in (ii) diagnosis of Zika virus infection. Thereby, use ofthe antibody, or an antigen binding fragment thereof, according to thepresent invention (and in particular its preferred embodiments asdescribed above) is preferred in (i) prevention and/or treatment of Zikavirus infection as described herein; or in (ii) diagnosis of Zika virusinfection as described herein.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be isolated from a subject, forexample an isolated tissue sample taken from, for example, nasalpassages, sinus cavities, salivary glands, lung, liver, pancreas,kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary,adrenals, thyroid, brain, skin or blood, preferably plasma or serum. Themethods of diagnosis may also include the detection of anantigen/antibody complex, in particular following the contacting of anantibody or an antibody fragment with a sample. Such a detection step istypically performed at the bench, i.e. without any contact to the humanor animal body. Examples of detection methods are well-known to theperson skilled in the art and include, e.g., ELISA (enzyme-linkedimmunosorbent assay).

Prevention of Zika virus infection refers in particular to prophylacticsettings, wherein the subject was not diagnosed with Zika virusinfection (either no diagnosis was performed or diagnosis results werenegative) and/or the subject does not show symptoms of Zika virusinfection. Accordingly, prevention of Zika virus infection includes“post-exposure prophylaxis” (PEP), i.e. preventive treatment after apossible Zika virus infection, for example after a mosquito bite in aZika virus affected area. Prevention of Zika virus infection is inparticular useful in high-risk subjects, such as in pregnant subjectsand/or in subjects staying in Zika virus affected areas (such assubjects living in Zika virus affected areas or travelling to Zika virusaffected areas).

In therapeutic settings, in contrast, the subject is typically infectedby Zika virus, diagnosed with Zika virus infection and/or showingsymptoms of Zika virus infection. Of note, the terms “treatment” and“therapy”/“therapeutic” of ZIKV infection include (complete) cure aswell as attenuation of ZIKV infection.

Preferred methods of diagnosis of Zika virus infection are the diagnosismethods as described herein, e.g. using the neutralizing antibody, orantigen binding fragment thereof, according to the present inventionand/or the ZIKV NS1-binding antibody, or antigen binding fragmentthereof, according to the present invention.

Accordingly, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is preferably used fortreatment of Zika virus infection in subjects diagnosed with Zika virusinfection or in subjects showing symptoms of Zika infection.

It is also preferred that the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention is used for preventionand/or treatment of Zika virus infection in asymptomatic subjects. Thosesubjects may be diagnosed or not diagnosed with Zika virus infection.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used for preventionand/or treatment of Zika virus infection in pregnant subjects, inparticular to prevent congenital infection. For example, this may beperformed in a similar manner as for the prevention of HCMV congentialinfection as described in Nigro G, Adler S P, La Torre R, Best A M,Congenital Cytomegalovirus Collaborating Group: Passive immunizationduring pregnancy for congenital cytomegalovirus infection; N Engl J Med2005, 353:1350-1362.

Without being bound to any theory, it is assumed that the antibody, orthe antigen-binding fragment thereof, according to the present inventioncan pass the placenta through the interaction with FcRn if administeredto a pregnant subject, e.g. by (i.v.) injection or any other route ofadministration as described herein. Importantly, the interaction of“LALA” variants of antibodies as described herein with FcRn is notcompromised. It is believed that FcRn is already expressed in the firsttrimester in the placenta.

Alternatively, the antibody, or the antigen-binding fragment thereof,according to the present invention may also be administered to theextra-amniotic space.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used for preventionand/or treatment of Zika virus infection, wherein the antibody, or theantigen binding fragment thereof, the nucleic acid, the vector, thecell, or the pharmaceutical composition is administered up to seven daysafter (a possible) Zika virus infection, preferably up to five daysafter (a possible) Zika virus infection, more preferably up to four daysafter (a possible) Zika virus infection, even more preferably up tothree days after (a possible) Zika virus infection, and most preferablyup to one day after (a possible) Zika virus infection. Such a treatmentschedule may be useful in therapeutic settings as well as inprophylactic settings, in particular in post-exposure prophylaxis (PEP).

In PEP typically the first administration of the the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention is as soon as possible after a possible ZIKV infection, e.g.after a mosquito bite in a ZIKV affected area. Accordingly, in PEP thefirst administration of the the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention is typically up to one ormore days after (a possible) ZIKV infection, as described above.

It is also preferred that the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention is used for preventionand/or treatment of Zika virus infection, wherein the antibody, or theantigen binding fragment thereof, the nucleic acid, the vector, thecell, or the pharmaceutical composition is administered up to threemonths before (a possible) Zika virus infection, preferably up to onemonth before (a possible) Zika virus infection, more preferably up totwo weeks before (a possible) Zika virus infection, even more preferablyup to one week before (a possible) Zika virus infection, and mostpreferably up to one day before (a possible) Zika virus infection. Sucha treatment schedule refers in particular to a prophylactic setting.

In general—and in particular in PEP—after the first administration ofthe antibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention, one or more subsequent administrations may follow,preferably a single dose per day or per every second day for 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 days. Itis also preferred that after the first administration of the antibody,or an antigen binding fragment thereof, according to the presentinvention, the nucleic acid according to the present invention, thevector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention, one or more subsequent administrations may follow,preferably a single dose once or twice per week for 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 weeks. It is alsopreferred that after the first administration of the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention, one or more subsequent administrations may follow, preferablya single dose every 2 or 4 weeks for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 weeks. It is also preferredthat after the first administration of the antibody, or an antigenbinding fragment thereof, according to the present invention, thenucleic acid according to the present invention, the vector according tothe present invention, the cell according to the present invention orthe pharmaceutical composition according to the present invention, oneor more subsequent administrations may follow, preferably a single doseevery two or four months for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,1, 15, 16, 17, 18, 19, 20, or 21 months. It is also preferred that afterthe first administration of the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention, one or more subsequentadministrations may follow, preferably a single dose once or twice peryear for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is administered at a(single) dose of 0.005 to 100 mg/kg bodyweight, preferably at a (single)dose of 0.0075 to 50 mg/kg bodyweight, more preferably at a (single)dose of 0.01 to 10 mg/kg bodyweight, even more preferably at a (single)dose of 0.05 to 5 mg/kg bodyweight, and particularly preferably at a(single) dose of 0.1 to 1 mg/kg bodyweight.

The antibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention may be administered by any number of routes such asoral, intravenous, intramuscular, intra-arterial, intramedullary,intraperitoneal, intrathecal, intraventricular, transdermal,transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual,intravaginal or rectal routes. Intravenous administration, orsubcutaneous administration or intramuscular administration arepreferred and intravenous administration or subcutaneous administrationare more preferred.

In pregnant subjects the antibody, or an antigen binding fragmentthereof, according to the present invention may also be administeredintra- or extra-amniotic, e.g. by injection.

Accordingly, the present invention also provides a method of preventingand/or treating Zika virus infection in a subject, wherein the methodcomprises administering to a subject in need thereof the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention. Preferred embodiments of this method correspond to preferredembodiments of the medical use as described above (and below, regardingcombination therapy). For example, a preferred subject in this method isa subject diagnosed with Zika virus infection or showing symptoms ofZika virus infection. Another preferred subject in this method is apregnant subject.

Combination Therapy

The administration of the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention in the methods and usesaccording to the invention can be carried out alone or in combinationwith a co-agent (also referred to as “additional active component”herein), which is in particular useful for preventing and/or treatingZIKV infection.

The invention encompasses the administration of the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention, wherein it is administered to a subject prior to,simultaneously or sequentially with other therapeutic regimens orco-agents useful for treating and/or preventing ZIKV infection. Saidantibody, nucleic acid, vector, cell or pharmaceutical composition, thatis administered simultaneously with said co-agents can be administeredin the same or different composition(s) and by the same or differentroute(s) of administration.

Said other therapeutic regimens or co-agents may be, for example, acheckpoint inhibitor.

Thus, in another aspect of the present invention the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention is administered in combination with a checkpoint inhibitor forthe (medical) uses as described herein.

Preferred checkpoint inhibitors are directed to a blockade of PD-1/PD-L1and/or of CTLA4 and, thus, include anti-PD-1 antibodies, anti-PD-L1antibodies and anti-CTLA4 antibodies. Thus, the pharmaceuticalcomposition according to the present invention may comprise one or moreof the additional active components.

It is also preferred that a ZIKV neutralizing antibody, or an antigenbinding fragment thereof, as described herein is combined with a ZIKVNS1-binding antibody, or an antigen binding fragment thereof, asdescribed herein as additional active component (co-agent). Thereby, thepathogenic role of NS1 may be blocked in addition to neutralization ofZIKV. Accordingly, a ZIKV NS1-binding antibody, or an antigen bindingfragment thereof, as described herein is a preferred additional activecomponent (co-agent).

The antibody, or the antigen binding fragment, according to the presentinvention can be present either in the same pharmaceutical compositionas the additional active component (co-agent) or, preferably, theantibody, or the antigen binding fragment, according to the presentinvention is comprised by a first pharmaceutical composition and theadditional active component (co-agent) is comprised by a secondpharmaceutical composition different from the first pharmaceuticalcomposition. Accordingly, if more than one additional active component(co-agent) is envisaged, each additional active component (co-agent) andthe antibody, or the antigen binding fragment, according to the presentinvention is preferably comprised by a different pharmaceuticalcomposition. Such different pharmaceutical compositions may beadministered either combined/simultaneously or at separate times or atseparate locations (e.g. separate parts of the body).

Preferably, the antibody, or the antigen binding fragment, according tothe present invention and the additional active component (co-agent)provide an additive therapeutic effect or, preferably, a synergistictherapeutic effect. The term “synergy” is used to describe a combinedeffect of two or more active agents that is greater than the sum of theindividual effects of each respective active agent. Thus, where thecombined effect of two or more agents results in “synergisticinhibition” of an activity or process, it is intended that theinhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Further Use and Kits

In a further aspect, the present invention also provides the use of theantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention for monitoring the quality of an anti-Zika vaccine bychecking that the antigen of said vaccine contains the specific epitopein the correct conformation. Preferred antigens comprised by such asanti-Zika vaccine to be checked include ZIKV envelope protein or anyother molecule/complex comprising or consisting of (i) domain III ofZIKV E protein (EDIII) as described above or (ii) a quaternary ZIKVepitope as described above.

Moreover, the present invention also provides the use of the antibody,or an antigen binding fragment thereof, according to the presentinvention, the nucleic acid according to the present invention, thevector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention in diagnosis of Zika virus infection.

In addition also the use of the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention in determining whether anisolated blood sample (e.g., whole blood, serum and/or plasma) isinfected with Zika virus is provided.

As described above, methods of diagnosis may include contacting anantibody or an antibody fragment with a sample. Such samples may beisolated from a subject, for example an isolated tissue sample takenfrom, for example, nasal passages, sinus cavities, salivary glands,lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract,heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood,preferably serum or plasma. The methods of diagnosis may also includethe detection of an antigen/antibody complex, in particular followingthe contacting of an antibody or an antibody fragment with a sample.Such a detection step is typically performed at the bench, i.e. withoutany contact to the human or animal body. Examples of detection methodsare well-known to the person skilled in the art and include, e.g., ELISA(enzyme-linked immunosorbent assay).

In a further aspect, the present invention also provides a kit of partscomprising at least one antibody, or antigen binding fragment thereof,according to the present invention, at least one nucleic acid accordingto the present invention, at least one vector according to the presentinvention, at least one cell according to the present invention, and/orat least one pharmaceutical composition according to the presentinvention. In addition, the kit may comprise means for administration ofthe antibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention, such as a syringe or a vessel, a leaflet, and/or aco-agent to be administered as described above.

Antibodies Specifically Binding to NS1 Protein of Zika Virus

In a further aspect, the present invention also provides an isolatedantibody, or an antigen binding fragment thereof, that specificallybinds to NS1 protein of ZIKA virus (ZIKV).

ZIKV NS1 protein (non-structural protein 1) occurs intracellular,secreted and cell-surface associated and in particular secreted ZIKV NS1protein is typically found in body fluids, such as serum, saliva, urineetc., of subjects infected with ZIKV. Secreted andcell-surface-associated NS1 are highly immunogenic and elicit productionof antibodies. NS1 is known to be an important biomarker for earlydiagnosis of ZIKV infection. Accordingly, the antibody, or an antigenbinding fragment thereof, according to the present invention thatspecifically binds to NS1 protein of ZIKA virus (ZIKV), is for exampleuseful in diagnosis of ZIKV infection.

In general, binding may be assessed by standard ELISA as known to theskilled person and as described above. Thereby, the relative affinitiesof antibody binding may be determined by measuring the concentration ofthe antibody (EC₅₀) required to achieve 50% maximal binding atsaturation. Preferably, the EC₅₀ of the antibody, or an antigen bindingfragment thereof, according to the present invention to ZIKV NS1 proteinis no more than 50 ng/ml, preferably said EC₅₀ is no more than 25 ng/ml,more preferably said EC₅₀ is no more than 15 ng/ml, even more preferablysaid EC₅₀ is no more than 10 ng/ml, and most preferably said EC₅₀ is nomore than 5 ng/ml, such as for example about 2 or 3 ng/ml.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, that specifically binds to NS1protein of ZIKA virus (ZIKV), does essentially not bind to Dengue virus(DENV) NS1 protein. Thereby “essentially not binding” means that for theantibody, or an antigen binding fragment thereof, no EC₅₀-value up to10² ng/ml, preferably up to 10³ ng/ml, more preferably up to 5*10³ng/ml, even more preferably up to 8*10³ ng/ml, and most preferably up to10⁴ ng/ml can be determined in a standard ELISA to Dengue virus (DENV)NS1 protein. In other words, the concentration of the antibody, or anantigen binding fragment thereof, required to achieve 50% maximalbinding at saturation (EC₅₀) to Dengue virus (DENV) NS protein in astandard ELISA is typically more than 10² ng/ml, preferably more than10³ ng/ml, more preferably more than 5*10³ ng/ml, even more preferablymore than 8*10³ ng/ml, and most preferably more than 10⁴ ng/ml.

It is also preferred that the antibody, or an antigen binding fragmentthereof, according to the present invention, that specifically binds toNS1 protein of ZIKA virus (ZIKV), does essentially not bind to Yellowfever virus (YFV) NS1 protein, West nile virus (WNV) NS1 protein,Japanese encephalitis virus (JEV) NS1 protein and/or to Tick-borneencephalitis virus (TBEV) NS1 protein. Thereby “essentially not binding”means that for the antibody, or an antigen binding fragment thereof, noEC₅₀-value up to 10² ng/ml, preferably up to 10³ ng/ml, more preferablyup to 5*10³ ng/ml, even more preferably up to 8*10³ ng/ml, and mostpreferably up to 10⁴ ng/ml can be determined in a standard ELISA toYellow fever virus (YFV) NS1 protein, West nile virus (WNV) NS1 protein,Japanese encephalitis virus (JEV) NS1 protein and/or to Tick-borneencephalitis virus (TBEV) NS1 protein. In other words, the concentrationof the antibody, or an antigen binding fragment thereof, required toachieve 50% maximal binding at saturation (EC₅₀) to Yellow fever virus(YFV) NS1 protein, West nile virus (WNV) NS1 protein, Japaneseencephalitis virus (JEV) NS1 protein and/or to Tick-borne encephalitisvirus (TBEV) NS1 protein in a standard ELISA is typically more than 10²ng/ml, preferably more than 10³ ng/ml, more preferably more than 5*10³ng/ml, even more preferably more than 8*10³ ng/ml, and most preferablymore than 10⁴ ng/ml.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, that specifically binds to NS1protein of ZIKA virus (ZIKV), is a human antibody. It is also preferredthat the antibody, or an antigen binding fragment thereof, according tothe present invention, that specifically binds to NS1 protein of ZIKAvirus (ZIKV), is a monoclonal antibody, preferably a human monoclonalantibody. Furthermore, it is also preferred that the antibody, or anantigen binding fragment thereof, according to the present invention,that specifically binds to NS1 protein of ZIKA virus (ZIKV), is arecombinant antibody.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, that specifically binds to NS1 proteinof ZIKA virus (ZIKV), comprises an Fc moiety. More preferably, theantibody according to the present invention, or an antigen bindingfragment thereof, that specifically binds to NS1 protein of ZIKA virus(ZIKV), comprises a CH2 L4A mutation, a CH2 L5A mutation, or both. For adetailed description of the antibody according to the present invention,or an antigen binding fragment thereof, comprising an Fc moiety and/orcomprises a CH2 L4A mutation, a CH2 L5A mutation, or both, it isreferred to the detailed description of the Fc moiety and of the CH2 L4Amutation, CH2 L5A mutation, or both in the context of the neutralizingantibodies according to the present invention as above. Thecorresponding detailed description as well as preferred embodimentsapply accordingly also for the antibody according to the presentinvention, or an antigen binding fragment thereof, that specificallybinds to NS1 protein of ZIKA virus (ZIKV).

However, it is also preferred that the antibody according to the presentinvention, or an antigen binding fragment thereof, that specificallybinds to NS1 protein of ZIKA virus (ZIKV), does not comprise an Fcmoiety. In particular it is preferred that the antibody according to thepresent invention, or an antigen binding fragment thereof, thatspecifically binds to NS1 protein of ZIKA virus (ZIKV), is a purifiedantibody, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv. Evenmore preferably, the antibody according to the present invention, or anantigen binding fragment thereof, that specifically binds to NS1 proteinof ZIKA virus (ZIKV), is labelled as described herein, for examplebiotinylated, such as a biotinylated Fab, Fab′, or F(ab′)2 fragment.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, that specifically binds to NS1 proteinof ZIKA virus (ZIKV), binds to antigenic site S1 and/or to antigenicsite S2 of Zika virus NS1 protein. The present inventors havesurprisingly found that anti-ZIKV NS1 antibodies binding to antigenicsite S1 and/or to antigenic site S2 of ZIKV NS1 protein are notcross-reactive with dengue virus NS1 protein (DENV NS1). Anti-ZIKV NS1antibodies, which bind neither to antigenic site S1 nor to antigenicsite S2 of ZIKV NS1 protein, in contrast, are typically cross-reactivewith DENV NS1. This surprising finding indicates that antigenic sites S1and S2 on ZIKV NS1 can be used to distinguish ZIKV NS1-specificantibodies from antibodies cross-reactive to DENV NS1.

Most preferably, the antibody according to the present invention, or anantigen binding fragment thereof, that specifically binds to NS1 proteinof ZIKA virus (ZIKV), binds to antigenic site S2 of Zika virus NS1protein. Antigenic site S2 is highly conserved in multiple ZIKVlineages, but it is not homologous in sequence and structure with thecorresponding site on NS1 of other flaviviruses, thereby providing aunique specificity for ZIKV.

Antigenic sites S1 and S2 of ZIKV NS1 protein were identified by thepresent inventors as described in Example 3, FIG. 6. Whether an antibodyis binding to antigenic site S1 and/or S2 may be easily identified bythe skilled person by using cross-competition studies, for example asdescribed below or in Example 3, wherein the S1-specific antibodyaccording to gZKA15 (SEQ ID NOs: 91-99) and/or the S2-specific antibodyaccording to gZKA35 (SEQ ID NOs: 127-135) may be used as “secondantibody”. In such a competition assay, presence of any competition(full or partial) with gZKA15 and/or gZKA35 indicates that the antibodyto be tested binds to antigenic site S1 and/or to antigenic site S2,respectively.

In general, for a competition assay commercially available systems forcharacterization of protein-protein binding, such as for example “Octet®RED96 System” provided by ForteBio, may be used, in particular accordingto the supplier's instructions.

In an exemplary competition assay, e.g. using “Octet® RED96 System”provided by ForteBio, ZIKV-NS1 protein (e.g. diluted to 2.5 μg/ml inPBS) may be immobilized (e.g. for 7-9 minutes) on the surface of an APScoated sensor-chip. Coated biosensors may then be placed into wellscontaining blocking buffer (e.g., 0.1% BSA in PBS; e.g. for 6 minutes)to block free Biosensor binding sites. Coated-Biosensors may then beincubated (e.g., for 8 minutes) with the antibody/antibodies to betested (e.g. diluted in blocking buffer for example at 10 μg/ml). Afterbinding of the antibodies to be tested (step 1), Biosensors were movedto wells containing the “second antibodies”, e.g. gZKA15 and/or gZKA35(e.g. for 8 minutes) (step 2). Competition, partial competition or nocompetition can thus be determined in step 2, depending on whether noassociation (competition), low association (partial competition) or(strong) association (no competition) is detected.

As described above, the antibody according to the present invention, orthe antigen binding fragment thereof, preferably comprises (at least)three complementarity determining regions (CDRs) on a heavy chain and(at least) three CDRs on a light chain. In general, complementaritydetermining regions (CDRs) are the hypervariable regions present inheavy chain variable domains and light chain variable domains.Typically, the CDRs of a heavy chain and the connected light chain of anantibody together form the antigen receptor. Usually, the three CDRs(CDR1, CDR2, and CDR3) are arranged non-consecutively in the variabledomain. Since antigen receptors are typically composed of two variabledomains (on two different polypeptide chains, i.e. heavy and lightchain), there are six CDRs for each antigen receptor (heavy chain:CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A singleantibody molecule usually has two antigen receptors and thereforecontains twelve CDRs. The CDRs on the heavy and/or light chain may beseparated by framework regions, whereby a framework region (FR) is aregion in the variable domain which is less “variable” than the CDR. Forexample, a chain (or each chain, respectively) may be composed of fourframework regions, separated by three CDR's.

The sequences of the heavy chains and light chains of five exemplaryantibodies of the invention, comprising three different CDRs on theheavy chain and three different CDRs on the light chain were determined.The position of the CDR amino acids are defined according to the IMGTnumbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al.(2009) Nucleic Acids Res. 37, D1006-D1012).

Table 4 shows the SEQ ID NO's of the amino acid sequences of the heavychain CDR's (CDRH1, CDRH2, and CDRH3) and of the heavy chain variableregion (referred to as “VH”) of exemplary antibodies according to thepresent invention:

Antibody name CDRH₁ CDRH₂ CDRH₃ VH ZKA₁₅ 91 92 93 98 ZKA₂₅ 109 110 111116 ZKA₃₅ 127 128 129 134 ZKA₁₀ 153 154 155 156 ZKA₁₈ 157 158 159 160ZKA₂₈ 161 162 163 164 ZKA₂₉ 165 166 167 168 ZKA₃₃ 169 170 171 172 ZKA₃₉173 174 175 176 ZKA₄₃ 177 178 179 180 ZKA₄₄ 181 182 183 184 ZKA₄₆ 185186 187 188 ZKA₅₀ 189 190 191 192 ZKA₅₄ 193 194 195 196 ZKB₁₈ 197 198199 200 ZKB₂₀ 201 202 203 204 ZKB₂₁ 205 206 207 208 ZKB₂₃ 209 210 211212 ZKC₂₉ 213 214 215 216 ZKC₃₁ 217 218 219 220 ZKC₃₂ 221 222 223 224ZKC₃₃ 225 226 227 228 ZKC₃₄ 229 230 231 232 ZKD₂₅ 233 234 235 236

Table 5 below shows the SEQ ID NO's of the amino acid sequences of thelight chain CDR's (CDRL1, CDRL2, and CDRL3) and of the light chainvariable region (referred to as “VL”) of exemplary antibodies accordingto the present invention:

Antibody name CDRL₁ CDRL₂ CDRL₂ long CDRL₃ VL ZKA₁₅ 94 95 96 97 99 ZKA₂₅112 113 114 115 117 ZKA₃₅ 130 131 132 133 135

It is thus preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises amino acidsequences having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to at least one of theCDR sequences, the VH sequence and/or the VL sequence shown in Table 4and/or in Table 5.

It is preferred that the antibody or antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to anyof SEQ ID NOs: 93, 111, 129, 155, 159, 163, 167, 171, 175, 179, 183,187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, and 235; ora functional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to anyof SEQ ID NOs: 93, 111 and 129, or a functional sequence variant thereofhaving at least 70%, at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity. Thereby, it ispreferred that at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 93 or a functional sequence variant thereof having at least 70%,at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity. It is also preferred that at least one CDR,preferably the at least one heavy chain CDRH3, comprises or consists ofan amino acid sequence according to SEQ ID NO: 111 or a functionalsequence variant thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity. Moreover, it is also preferred that at least one CDR,preferably the at least one heavy chain CDRH3, comprises or consists ofan amino acid sequence according to SEQ ID NO: 129 or a functionalsequence variant thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 129, or a functional sequence variant thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity.

It is also preferred, that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to any of SEQ ID NOs: 91, 109, 127, 153, 157,    161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209,    213, 217, 221, 225, 229, and 233; or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to any of SEQ ID NOs: 92, 110, 128, 154, 158, 162, 166,    170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218,    222, 226, 230, and 234; or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to any of SEQ ID NOs: 93, 111, 129, 155, 159,    163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211,    215, 219, 223, 227, 231, and 235; or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity.

Still more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to any of SEQ ID NOs: 91, 109 and 127, or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to any of SEQ ID NOs: 92, 110 and 128, or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to any of SEQ ID NOs: 93, 111 and 129, or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 91 or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 92 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NOs: 93 or a functional sequence    variant thereof having at least 70%, at least 75%, at least 80%, at    least 85%, at least 88%, at least 90%, at least 92%, at least 95%,    at least 96%, at least 97%, at least 98% or at least 99% sequence    identity.

It is also even more preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain comprising at least one CDRH1, at least one CDRH2 and at least oneCDRH3 and a light chain comprising at least one CDRL1, at least oneCDRL2 and at least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 109 or a functional sequence    variant thereof having at least 70%, at least 75%, at least 80%, at    least 85%, at least 88%, at least 90%, at least 92%, at least 95%,    at least 96%, at least 97%, at least 98% or at least 99% sequence    identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 110 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NOs: 111 or a functional sequence    variant thereof having at least 70%, at least 75%, at least 80%, at    least 85%, at least 88%, at least 90%, at least 92%, at least 95%,    at least 96%, at least 97%, at least 98% or at least 99% sequence    identity.

It is particularly preferred, that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain comprising at least one CDRH1, at least one CDRH2 and at least oneCDRH3 and a light chain comprising at least one CDRL1, at least oneCDRL2 and at least one CDRL3, wherein

-   (i) the at least one heavy chain CDRH1 comprises an amino acid    sequence according to SEQ ID NO: 127 or a functional sequence    variant thereof having at least 70%, at least 75%, at least 80%, at    least 85%, at least 88%, at least 90%, at least 92%, at least 95%,    at least 96%, at least 97%, at least 98% or at least 99% sequence    identity;-   (ii) the at least one CDRH2 comprises an amino acid sequence    according to SEQ ID NO: 128 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity; and/or-   (iii) the at least one heavy chain CDRH3 comprises an amino acid    sequence according to SEQ ID NOs: 129 or a functional sequence    variant thereof having at least 70%, at least 75%, at least 80%, at    least 85%, at least 88%, at least 90%, at least 92%, at least 95%,    at least 96%, at least 97%, at least 98% or at least 99% sequence    identity.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to any of SEQ ID NOs: 94, 112 and 130, or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to any of SEQ ID NOs: 95, 96, 113, 114, 131 and 132, or a    functional sequence variant thereof having at least 70%, at least    75%, at least 80%, at least 85%, at least 88%, at least 90%, at    least 92%, at least 95%, at least 96%, at least 97%, at least 98% or    at least 99% sequence identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to any of SEQ ID NOs: 97, 115 and 133, or a functional    sequence variant thereof having at least 70%, at least 75%, at least    80%, at least 85%, at least 88%, at least 90%, at least 92%, at    least 95%, at least 96%, at least 97%, at least 98% or at least 99%    sequence identity.

Even more preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 94 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to SEQ ID NO: 95 or 96, or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 97 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity.

It is also even more preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain comprising at least one CDRH1, at least one CDRH2 and at least oneCDRH3 and a light chain comprising at least one CDRL1, at least oneCDRL2 and at least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 112 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to SEQ ID NO: 113 or 114, or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 115 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity.

It is particularly preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain comprising at least one CDRH1, at least one CDRH2 and at least oneCDRH3 and a light chain comprising at least one CDRL1, at least oneCDRL2 and at least one CDRL3, wherein

-   (i) the at least one CDRL1 comprises an amino acid sequence    according to SEQ ID NO: 130 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity;-   (ii) the at least one CDRL2 comprises an amino acid sequence    according to SEQ ID NO: 131 or 132, or a functional sequence variant    thereof having at least 70%, at least 75%, at least 80%, at least    85%, at least 88%, at least 90%, at least 92%, at least 95%, at    least 96%, at least 97%, at least 98% or at least 99% sequence    identity; and/or-   (iii) the at least one CDRL3 amino comprises an amino acid sequence    according to SEQ ID NO: 133 or a functional sequence variant thereof    having at least 70%, at least 75%, at least 80%, at least 85%, at    least 88%, at least 90%, at least 92%, at least 95%, at least 96%,    at least 97%, at least 98% or at least 99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises CDRH1, CDRH2, and CDRH3amino acid sequences (i) according to SEQ ID NOs: 91-93; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (ii) according to SEQ ID NOs: 109-111; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(iii) according to SEQ ID NOs: 127-129; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (iv)according to SEQ ID NOs: 153-155; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (v) accordingto SEQ ID NOs: 157-159; or functional sequence variants thereof havingat least 70%, at least 75%, at least 80%, at least 85%, at least 88%, atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity; (vi) according to SEQ IDNOs: 161-163; or functional sequence variants thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity; (vii) according to SEQ ID NOs:165-167; or functional sequence variants thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity; (viii) according to SEQ ID NOs: 169-171; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (ix) according to SEQ ID NOs: 173-175; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (x) according to SEQ ID NOs: 177-179; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;(xi) according to SEQ ID NOs: 181-183; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xii)according to SEQ ID NOs: 185-187; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xiii)according to SEQ ID NOs: 189-191; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xiv)according to SEQ ID NOs: 193-195; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xv)according to SEQ ID NOs: 197-199; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xvi)according to SEQ ID NOs: 201-203; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xvii)according to SEQ ID NOs: 205-207; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xviii)according to SEQ ID NOs: 209-211; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xix)according to SEQ ID NOs: 213-215; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xx)according to SEQ ID NOs: 217-219; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxi)according to SEQ ID NOs: 221-223; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxii)according to SEQ ID NOs: 225-227; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (xxiii)according to SEQ ID NOs: 229-231; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; or (xxiv)according to SEQ ID NOs: 233-235; or functional sequence variantsthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

It is particularly preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises CDRH1,CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 aminoacid sequences (i) according to SEQ ID NOs: 91-95 and 97; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (ii) according to SEQ ID NOs: 91-94 and 96-97; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (iii) according to SEQ ID NOs: 109-113 and 115; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity; (iv) according to SEQ ID NOs: 109-112 and 114-115; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; (v) according to SEQ ID NOs: 127-131 and 133; orfunctional sequence variants thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity; or (vi) according to SEQ ID NOs: 127-130 and 132-133;or functional sequence variants thereof having at least 70%, at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises CDRH1, CDRH2, and CDRH3amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences(i) according to SEQ ID NOs: 127-131 and 133; or functional sequencevariants thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;or (ii) according to SEQ ID NOs: 127-130 and 132-133; or functionalsequence variants thereof having at least 70%, at least 75%, at least80%, at least 85%, at least 88%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chainvariable region (VH) and, optionally, a light chain variable region(VL), wherein the heavy chain variable region (VH) comprises or consistsof an amino acid sequence according to any of SEQ ID NOs: 98, 116, 134,156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208,212, 216, 220, 224, 228, 232, and 236; or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises (i) a heavy chain variableregion (VH) amino acid sequence according to SEQ ID NO: 98 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 99 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity; (ii) a heavychain variable region (VH) amino acid sequence according to SEQ ID NO:116 or a functional sequence variant thereof having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity and/or a light chain variable region (VL)amino acid sequence according to SEQ ID NO: 117 or a functional sequencevariant thereof having at least 70%, at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identity;or (iii) a heavy chain variable region (VH) amino acid sequenceaccording to SEQ ID NO: 134 or a functional sequence variant thereofhaving at least 70%, at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity and/or a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 135 ora functional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity.

Most preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain variableregion (VH) amino acid sequence according to SEQ ID NO: 134 or afunctional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity and/or a light chain variable region (VL) amino acidsequence according to SEQ ID NO: 135 or a functional sequence variantthereof having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention is gZKA15, gZKA25, or gZKA35, morepreferably the antibody, or the antigen binding fragment thereof, isgZKA25 or gZKA35, even more preferably the antibody, or the antigenbinding fragment thereof, is gZKA35.

The present inventors have isolated monoclonal antibody (mAb) accordingto the present invention, which are referred to herein as ZKA15, ZKA25and ZKA35 (cf. Tables 4 and 5, Example 1). Based on those antibodies, inparticular on the VH and VL genes of those antibodies, the terms“gZKA15”, “gZKA25” and “gZKA35”, as used herein, refer to the respective“generic” antibodies, or antigen binding fragments thereof.

Namely, “gZKA15” refers to an antibody, or antigen binding fragmentthereof, having a CDRH1 amino acid sequence according to SEQ ID NO: 91,a CDRH2 amino acid sequence according to SEQ ID NO: 92, a CDRH3 aminoacid sequence according to SEQ ID NO: 93, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 94, a CDRL2 amino acid sequence according to SEQID NO: 95 or 96, and a CDRL3 amino acid sequence according to SEQ ID NO:97. The heavy chain variable region (V_(H)) has preferably an amino acidsequence according to SEQ ID NO: 98 and the light chain variable region(V_(L)) has preferably an amino acid sequence according to SEQ ID NO:99.

“gZKA25” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 109, a CDRH2amino acid sequence according to SEQ ID NO: 110, a CDRH3 amino acidsequence according to SEQ ID NO: 111, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 112, a CDRL2 amino acid sequence according toSEQ ID NO: 113 or 114, and a CDRL3 amino acid sequence according to SEQID NO: 115. The heavy chain variable region (V_(H)) has preferably anamino acid sequence according to SEQ ID NO: 116 and the light chainvariable region (V_(L)) has preferably an amino acid sequence accordingto SEQ ID NO: 117.

“gZKA35” refers to an antibody, or antigen binding fragment thereof,having a CDRH1 amino acid sequence according to SEQ ID NO: 127, a CDRH2amino acid sequence according to SEQ ID NO: 128, a CDRH3 amino acidsequence according to SEQ ID NO: 129, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 130, a CDRL2 amino acid sequence according toSEQ ID NO: 131 or 132, and a CDRL3 amino acid sequence according to SEQID NO: 133. The heavy chain variable region (V_(H)) has preferably anamino acid sequence according to SEQ ID NO: 134 and the light chainvariable region (V_(L)) has preferably an amino acid sequence accordingto SEQ ID NO: 135.

The detailed description above referring to “production of antibodies”(above section “production of antibodies”) and “optional additionalfeatures of antibodies” (above section “optional additional features ofantibodies”) apply to all antibodies, and antigen binding fragmentsthereof, as described in the present application—i.e. those sectionsapply not only to the neutralizing antibodies, and antigen-bindingfragments thereof, according to the present invention, but also to theNS1-protein binding antibodies, and antigen-binding fragments thereof,according to the present invention.

In particular, it is preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention is labelled, forexample biotinylated.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention is biotinylated.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention is conjugated to an enzyme,such as horseradish peroxidase (HRP). Conjugation of antibodies to HRPare, for example, described in Wisdom G B. Conjugation of antibodies tohorseradish peroxidase. Methods Mol Biol. 2005; 295:127-30 or inAntibodies—a laboratory manual. Edited by Edward A. Greenfield, Secondedition 2012, Cold Spring Harbor Laboratory Press, ISBN: 9781936113811.

For example, antibodies of the invention, or the antigen bindingfragments thereof, may be coupled to a detectable label, for example toprovide measurability, e.g. for quantification or to facilitate imaging.Labeled antibodies may be employed in a wide variety of assays, inparticular in immunoassays, employing a wide variety of labels.Preferred labels include radionuclides, enzymes, coenzymes, fluorescers,chemiluminescers, chromogens, enzyme substrates or co-factors, enzymeinhibitors, prosthetic group complexes, free radicals, particles, dyes(e.g., fluorescent dyes, tandem dyes), and the like. Examples ofsuitable enzymes include horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 1251, 1311, 35S, or 3H. Suchlabeled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like, preferably in ELISA. Labeled antibodiesaccording to the present invention may be thus be used in such assaysfor example as described in U.S. Pat. Nos. 3,766,162; 3,791,932;3,817,837; and 4,233,402.

Preferred labels include (i) enzymes as described above, e.g.horseradish peroxidase (HRP) or alkaline phosphatase, in particular inBlockade-of-binding assay, Western Blotting, ELISA andimmunohistochemistry; (ii) prosthetic group complexes as describedabove, e.g. streptavidin/biotin and avidin/biotin, in particular inELISA and immunohistochemistry; (iii) fluorescers as described above,such as fluorescent dyes and fluorescent proteins (e.g., (enhanced)green fluorescent protein (EGFP); TagBFP, Turquoise, Venus, KO2, Cherry,Apple, Kate2), in particular in immunofluorescence and flow cytometry;and (iv) tandem dyes in flow cytometry.

Preferably, the antibody, or the antigen binding fragment thereof, isbiotinylated. Biotinylation is rapid, specific and is unlikely toperturb the natural function of the molecule due to the small size ofbiotin (MW=244.31 g/mol). Biotin binds to streptavidin and avidin withan extremely high affinity, fast on-rate, and high specificity.Biotin-binding to streptavidin and avidin is resistant to extremes ofheat, pH and proteolysis, making capture of biotinylated moleculespossible in a wide variety of environments. The antibody, or the antigenbinding fragment thereof, according to the present invention may bebiotinylated chemically or enzymatically. Chemical biotinylationutilizes various conjugation chemistries to yield nonspecificbiotinylation of amines (e.g., NHS-coupling gives biotinylation of anyprimary amines in the antibody, see below). Enzymatic biotinylationresults in biotinylation of a specific lysine within a certain sequenceby use of a bacterial biotin ligase.

Moreover, a second antibody, or antibody fragment thereof, may also beused as label. In this case, the antibody, or the antigen bindingfragment thereof, according to the present invention is conjugated to asecond antibody, or antibody fragment thereof, to form an antibodyheteroconjugate as described for example in U.S. Pat. No. 4,676,980. Inthis case, the second antibody may optionally be labelled as describedherein.

Methods for coupling antibodies to labels are well known in the art. Forexample, in the antibody or in the antigen binding fragment thereof theside chain of lysine, which terminates in a primary amine (—NH₂), may beused to link labels covalently to the antibody or in the antigen bindingfragment thereof. Many variant labeling procedures are described in theliterature. For example, the labelling approach may be selected from thegroup consisting of NHS esters, heterobifunctional reagents,carbodiimides and sodium periodate.

NHS esters may be used in particular in the case of fluorescent dyelabels. A fluorescent dye label may be purchased in an activated form ofthe label with an inbuilt NHS ester (also called a ‘succinimidylester’). The activated dye can be reacted under appropriate conditionswith the antibody or with the antigen binding fragment thereof (e.g. viaa lysine group). Excess reactive dye can be removed (e.g. by columnchromatography) before the labeled antibody or antigen binding fragmentthereof can be used in an immunoassay.

Heterobifunctional reagents may be used in particular if the label is aprotein molecule (e.g. HRP, alkaline phosphatase, or phycoerythrin). Inthis case, the antibody or the antigen binding fragment thereof and thelabel may have multiple amines. In this situation some of the lysines onone molecule (e.g. on the antibody or on the antigen binding fragmentthereof) may be modified to create a new reactive group (X) and lysineson the label to create another reactive group (Y) (or vice versa). A‘heterobifunctional reagent’ is then used to introduce the Y groups,which subsequently react with X groups when antibody and label aremixed, thus creating heterodimeric conjugates.

Carbodiimides, such as EDC, may be used in particular to create covalentlinks between amine- and carboxyl-containing molecules. Carbodiimidesactivate carboxyl groups, and the activated intermediate is thenattacked by an amine (e.g. provided by a lysine residue on the antibodyor the antigen binding fragment thereof). Carbodiimides may be used inparticular to conjugate antibodies to carboxylated particles (e.g. latexparticles, magnetic beads), and to other carboxylated surfaces, such asmicrowell plates or chip surfaces. Carbodiimides may also be used toattach dyes or protein labels to antibodies or antigen binding fragmentsthereof.

Sodium periodate may be used in particular for labelling withhorseradish peroxidase (HRP). Periodate activates carbohydrate chains onthe HRP molecule to create aldehyde groups, which are capable ofreacting with lysines on the antibody or antigen binding fragmentthereof. Since HRP itself has very few lysines, it is relatively easy tocreate antibody-HRP conjugates without significant HRP polymerization.

Optionally, linkers may be used between the labels and the antibodies ofthe invention, e.g., as described in U.S. Pat. No. 4,831,175. Antibodiesor, antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art, e.g., as described in U.S. Pat. No. 5,595,721.

Accordingly, the present invention also provides a complex comprising

-   (i) the antibody, or the antigen binding fragment thereof, according    to the present invention; and-   (ii) a label as described above.

Accordingly, such a complex is preferably a label conjugated to theantibody, or the antigen binding fragment thereof, according to thepresent invention. Preferably, the label and the antibody, or theantigen binding fragment thereof, according to the present invention arecovalently linked.

For example, the complex according to the present invention may be afusion protein comprising (i) the antibody according to the presentinvention and (ii) a label, which is a peptide or a protein, such as afluorescent peptide or protein, e.g. EGFP.

In another aspect, the present invention also provides a nucleic acidmolecule comprising a polynucleotide encoding the antibody, or theantigen binding fragment thereof, according to the present invention asdescribed above or the complex according to the present invention asdescribed above, in particular if the complex according to the presentinvention is a fusion protein as described above.

Examples of nucleic acid molecules and/or polynucleotides include, e.g.,a recombinant polynucleotide, a vector, an oligonucleotide, an RNAmolecule such as an rRNA, an mRNA, an miRNA, an siRNA, or a tRNA, or aDNA molecule such as a cDNA.

Nucleic acid sequences encoding part or all of the light and heavychains and CDRs of the antibodies of the present invention arepreferred. Preferably provided herein are thus nucleic acid sequencesencoding part or all of the light and heavy chains, in particular VH andVL sequences and CDRs of the exemplary antibodies of the invention.Tables 4 and 5 provide the SEQ ID numbers for the amino acid sequencesof the CDRs and VH and VL of exemplary antibodies according to thepresent invention.

Table 6 below provides the SEQ ID numbers for exemplary nucleic acidsequences encoding the CDRs and VH and VL of exemplary antibodiesaccording to the present invention. Due to the redundancy of the geneticcode, the present invention also comprises sequence variants of thesenucleic acid sequences and in particular such sequence variants, whichencode the same amino acid sequences.

A nucleic acid molecule is a molecule comprising, preferably consistingof nucleic acid components. The term nucleic acid molecule preferablyrefers to DNA or RNA molecules. In particular, it is used synonymouswith the term “polynucleotide”. Preferably, a nucleic acid molecule is apolymer comprising or consisting of nucleotide monomers which arecovalently linked to each other by phosphodiester-bonds of asugar/phosphate-backbone. The term “nucleic acid molecule” alsoencompasses modified nucleic acid molecules, such as base-modified,sugar-modified or backbone-modified etc. DNA or RNA molecules.

Table 6 shows exemplary nucleic acid sequences of the CDR's and theheavy chain variable region (VH) and the light chain variable region(VL) of three exemplary antibodies according to the present invention(“ZKA15”, “ZKA25”, “ZKA35”):

SEQ ID NO. Nucleic acid sequence ZKA15 CDRH₁ 100ggtggcttcatcaatagttactac CDRH₂ 101 atctataaaagtgggagcacc CDRH₃ 102gcgagagatccctacggtgactacgttaaggcttttgatat t CDRL₁ 103cagagcctcctgcatagtaatggatacaactat CDRL₂ 104 ttgggttct CDRL₂ 105ctgatctatttgggttctaatcgggcc long CDRL₃ 106 atgcaagctctacaaactgtcact VH107 caggtgcagctgcaggagtcggggccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctccggtggcttcatcaatagttactactggagctggatccggcagcccgccgggaagggactggagtggattgggcgtatctataaaagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatgtcactagacacgtccaagtaccagttctccctgaagctgaggtctgtgaccgccgctgacacggccgtgtattactgtgcgagagatccctacggtgactacgttaaggcttttgatatttggggccaagggacaatggtcaccgtctcttcag VL 108gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtaatggatacaactatttgaattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactgtcactttcggccctgggaccaaagtggata tcaaac ZKA25 CDRH₁ 118ggattcacctttagaagtcattgg CDRH₂ 119 ataaaggaagatggatatgagaaa CDRH₃ 120gcgagagatttgagggtatatagtgggagaggtttcgaccc c CDRL₁ 121 aaattgggggataaatatCDRL₂ 122 caagatagc CDRL₂ 123 gtcatctatcaagatagcaagcggccc long CDRL₃ 124caggcgtgggacagcagcactgtggta VH 125gaggtgcagttggtggagtctgggggaggcttggtccggcctggggggtccctgagactctcctgtgcagcctctggattcacctttagaagtcattggatgagttgggtccgccaggctccagggaaggggctggagtgggtggccaacataaaggaagatggatatgagaaatactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaagagcctgagagccgaggacacggccgtgtattactgtgcgagagatttgagggtatatagtgggagaggtttcgacccctggggccagggaaccctggtcaccgtctcctcag VL 126tcctatgagctgactcagccaccctcactgtccgtgtccccaggacagacagccagcatcacctgctctggagataaattgggggataaatatgcttgctggtatcagcagaagccaggccagtcccctgtgttggtcatctatcaagatagcaagcggccctcagggatccctgcgcgattctctggctccaactctgggaacacagccactctgaccatcagcgggacccaggctatggatgaggctgactattactgtcaggcgtgggacagcagcactgtggtattcggtggagggaccaagctgaccgtcctag ZKA35 CDRH₁ 136ggtggctccatcagcactggtggttactac CDRH₂ 137 atctattacagtgggaacacc CDRH₃ 138gcgaaaggaggagggagggagcgaccctttgactac CDRL₁ 139 agctccaacatcggaagaaattatCDRL₂ 140 aggaataat CDRL₂ 141 ctcatctataggaataatcagcggccc long CDRL₃ 142gtagcatgggatgacagccggagtggttttgtggta VH 143caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcactggtggttactactggagctggatccgccagcacccagggaagggcctggagtggattggttacatctattacagtgggaacacctactacaacccgtccctcaagagtcgagttaccatatcagttgacacctctaagaagcagttctccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgaaaggaggagggagggagcgaccctttgactactggggccagggaaccctggtcaccgtctcctcag VL 144cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagaaattatgtagactggtaccagcaactcccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgagcgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgtagcatgggatgacagccggagtggttttgtggtattcggcggagggaccaaggtgaccg tcctag

Preferably, the sequence of the nucleic acid molecule according to thepresent invention comprises or consists of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 100-108, 118-126, and 136-144.

It is also preferred that nucleic acid sequences according to theinvention include nucleic acid sequences having at least 70%, at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identity to the nucleic acid encoding a CDR, a VH sequence and/or aVL sequence used in an (exemplary) antibody according to the presentinvention, for example to the sequences shown in Table 6.

In general, the nucleic acid molecule may be manipulated to insert,delete or alter certain nucleic acid sequences. Changes from suchmanipulation include, but are not limited to, changes to introducerestriction sites, to amend codon usage, to add or optimizetranscription and/or translation regulatory sequences, etc. It is alsopossible to change the nucleic acid to alter the encoded amino acids.For example, it may be useful to introduce one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/orinsertions into the antibody's amino acid sequence. Such point mutationscan modify effector functions, antigen-binding affinity,post-translational modifications, immunogenicity, etc., can introduceamino acids for the attachment of covalent groups (e.g., labels) or canintroduce tags (e.g., for purification purposes). Mutations can beintroduced in specific sites or can be introduced at random, followed byselection (e.g., molecular evolution). For instance, one or more nucleicacids encoding any of the CDR regions, a VH sequence and/or a VLsequence of an (exemplary) antibody of the invention can be randomly ordirectionally mutated to introduce different properties in the encodedamino acids. Such changes can be the result of an iterative processwherein initial changes are retained and new changes at other nucleotidepositions are introduced. Further, changes achieved in independent stepsmay be combined. Different properties introduced into the encoded aminoacids may include, but are not limited to, enhanced affinity.

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid moleculeaccording to the present invention. Preferably, a vector comprises anucleic acid molecule as described above.

The term “vector” refers to a nucleic acid molecule, preferably to arecombinant nucleic acid molecule, i.e. a nucleic acid molecule whichdoes not occur in nature. A vector in the context of the presentinvention is suitable for incorporating or harboring a desired nucleicacid sequence. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule. Thus,the vector may comprise a sequence corresponding, e.g., to a desiredantibody or antibody fragment thereof according to the presentinvention. An expression vector may be used for production of expressionproducts such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.For example, an expression vector may comprise sequences needed fortranscription of a sequence stretch of the vector, such as a promotersequence. A cloning vector is typically a vector that contains a cloningsite, which may be used to incorporate nucleic acid sequences into thevector. A cloning vector may be, e.g., a plasmid vector or abacteriophage vector. A transfer vector may be a vector which issuitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors.

A vector in the context of the present invention may be, e.g., an RNAvector or a DNA vector.

Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector.

In a further aspect, the present invention also provides cell expressingthe antibody, or the antigen binding fragment thereof, according to thepresent invention; and/or comprising the vector according the presentinvention.

Examples of such cells include but are not limited to, eukaryotic cells,e.g., yeast cells, animal cells or plant cells. Preferably, the cellsare mammalian cells, more preferably a mammalian cell line. Preferredexamples include human cells, CHO cells, HEK293T cells, PER.C6 cells,NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, the cell may be transfected with a vector according tothe present invention, preferably with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably intoeukaryotic cells. In the context of the present invention, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, preferably intoeukaryotic cells, such as into mammalian cells. Such methods encompass,for example, electroporation, lipofection, e.g. based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine etc.Preferably, the introduction is non-viral.

Moreover, the cells of the present invention may be transfected stablyor transiently with the vector according to the present invention, e.g.for expressing the antibody, or the antigen binding fragment thereof,according to the present invention. Preferably, the cells are stablytransfected with the vector according to the present invention encodingthe antibody, or the antigen binding fragment thereof, according to thepresent invention. Alternatively, it is also preferred that the cellsare transiently transfected with the vector according to the presentinvention encoding the antibody, or the antigen binding fragmentthereof, according to the present invention.

In a further aspect, the present invention also provides a compositioncomprising the antibody, or the antigen binding fragment thereof,according to the present invention; the complex according to the presentinvention as described above; the nucleic acid molecule according to thepresent invention as described above; the vector according to thepresent invention as described above; or the cell according to thepresent invention as described above. A composition comprising theantibody, or the antigen binding fragment thereof, according to thepresent invention or the complex according to the present invention asdescribed above is preferred.

Such a composition may be a pharmaceutical composition as describedabove in the context of the neutralizing antibodies, whereby thedetailed description and preferred embodiments of such a pharmaceuticalcomposition as described above apply accordingly to the antibody, or theantigen binding fragment thereof, according to the present invention,that binds to ZIKV NS1 protein. However, the composition may also beused for non-pharmaceutical purposes, such as in diagnosis (of ZIKVinfection) or for analytical purposes.

Preferably, the composition is in liquid form, e.g. to provide theantibody or the antigen binding fragment thereof in a liquid for directuse, e.g. in a diagnosis assay. The liquid (vehicle) may be chosenaccording to the purpose, e.g. depending on the assay. Preferably, thecomposition according to the present invention comprises PBS(phosphate-buffered saline) or another buffer. Such buffers arepreferably biological buffers, and the composition may thus comprise anyof MES, BIS-TRIS, ADA, PIPES, ACES, MOPSO, BIS-TRIS propane, BES, MOPS,TES, HEPES, DIPSO, TAPSO, Trizma, POPSO, HEPPS, TRICINE, Gly-Gly,BICINE, HEPBS, TAPS, AMPD, AMPSO, CHES, CAPSO, AMP, CAPS and CABS. It isalso preferred that the composition comprises Ringer's solution. Inaddition, the composition may also comprise Tris, e.g., Tris-HCl.

The composition according to the present invention may also comprise adetergent e.g., a Tween (polysorbate), such as Tween 20 or Tween 80.Detergents are preferably present at low levels e.g., less than 0.01%.Compositions may also include sodium salts (e.g., sodium chloride) togive tonicity. For example, a concentration of 10±2 mg/ml NaCl istypical.

In addition, the composition according to the present invention mayoptionally comprise a protein stabilizer, such as BSA (bovine serumalbumin) or HSA (human serum albumin). Further examples of proteinstabilizers, which may optionally be included in the compositionaccording to the present invention, include buffers, e.g. as describedabove; salts, such as sodium chloride; amino acids, such as histidine,glycine, and arginine; polyols/disaccharides/polysaccharides, such astrehalose and sucrose (disaccharides), mannitol and sorbitol (sugaralcohols); surfactants, such as polysorbate 20, polysorbate 80, andproteins like HSA or BSA; polymers, such as dextran and polyethyleneglycol; and antioxidants.

Furthermore, the composition according to the present invention mayoptionally comprise a preservative, such as sodium azide. Preservativesare typically used to prevent microbial contamination.

In a further aspect, the present invention also provides a kit of partscomprising the antibody, or the antigen binding fragment thereof,according to the present invention, the complex according to the presentinvention or the composition according to the present invention.

Such a kit of parts may optionally further comprise one or more of thefollowing:

-   (i) one or more solutions, e.g. to be used in a diagnosis assay,    e.g. to dilute the antibody or the antigen binding fragment thereof;-   (ii) a leaflet, e.g. with instructions to use;-   (iii) a label as described above and, optionally, solutions and/or    further components required for labeling; and/or-   (iv) vessels or devices, e.g. useful in a diagnosis assay, for    example one or more ELISA plates.

Preferably, the kit according to the present invention as describedabove also comprises a substrate for the development of the color.Examples of such a substrate include p-NPP, in particular in case ofdetection through alkaline phosphatase; or an enzyme like ABTS, TMB orOPD, in particular in case of use of horse-radish peroxidase (HRP).Optionally, the substrate may be diluted in an appropriate buffer, e.g.a buffer as described above in the context of the composition accordingto the present invention. Alternatively, the substrate and the buffermay be provided as separate entities in the kit.

With regard to the label, the kit according to the present invention mayalso comprise an enzyme conjugated streptavidin, or another system todetect the binding of the probe antibody. For example, the probeantibody may be made in murinized form and in this case the binding maybe detected with an anti-mouse secondary antibody—without the need forbiotinylation. The anti-mouse secondary antibody is typically polyclonaland/or cross-adsorbed for not reacting with human antibodies.

Moreover, the kit according to the present invention preferablycomprises one or more ELISA plates. More preferably, those ELISA platesare pre-coated with ZIKV-NS1 protein. Optionally, such pre-coatedELISA-plates may be pre-blocked.

Diagnosis of Zika Virus Infection

In a further aspect, the present invention also provides the use of theantibody, or the antigen binding fragment thereof, according to thepresent invention, that binds to ZIKV NS1 protein, the complex accordingto the present invention, the composition according to the presentinvention or the kit of parts according to the present invention indiagnosis of Zika virus (ZIKV) infection.

Diagnosis of Zika virus (ZIKV) infection is typically performed invitro, e.g. in an isolated sample of the subject to be diagnosed.Preferred isolated samples of the subject include samples of a bodyfluid and tissue samples. A sample of a body fluid is more preferred.Preferred body fluids for diagnosis of ZIKV infection include blood(e.g. whole blood, plasma, serum), saliva and urine. Blood, inparticular plasma or serum, is most preferred.

Accordingly, the present invention also provides the use of theantibody, or the antigen binding fragment thereof, according to thepresent invention, that binds to ZIKV NS1 protein, the complex accordingto the present invention, the composition according to the presentinvention or the kit of parts according to the present invention indetermining whether an isolated sample (of a body fluid), such as anisolated blood sample, is infected with Zika virus. As described above,preferred body fluids for diagnosis of ZIKV infection include blood(e.g. whole blood, plasma, serum), saliva and urine. Blood, inparticular plasma or serum, is more preferred.

For diagnosis of Zika virus infection, different diagnosis assays may beused. Preferred diagnosis assays are immunoassays. Preferred examples ofimmunoassays include ELISA, immunofluorescence, immunohistochemistry andflow cytometry. Preferably, diagnosis includes ELISA. For example, astandard ELISA, a sandwich ELISA or a blockade of binding assay may beused.

Preferably, the diagnosis assay detects

-   (i) (the presence of) ZIKV NS1 protein itself; and/or-   (ii) (the presence of) anti-ZIKV NS1 antibodies-   in an (isolated) sample of a subject to be diagnosed.

Preferably, a blockade-of-binding assay is used. In this assay, anisolated sample from a subject to be diagnosed (e.g., a sample of a bodyfluid, such as blood (e.g. whole blood, plasma, serum), saliva andurine) is added to an ELISA plate coated with ZIKV NS1 protein andincubated (for example, for at least about 30 min or at least about onehour) to allow for binding. Thereafter, the antibody, or the antigenbinding fragment thereof, according to the present invention is added(as “probe antibody”), wherein the antibody or the antigen bindingfragment thereof according to the present invention is preferablylabelled, e.g. biotinylated or conjugated to horseradish peroxidase(HRP). After another incubation time (e.g., at least about 1 min,preferably at least about 3 min, more preferably at least about 5 min,even more preferably at least about 10 min, most preferably at leastabout 15 min), inhibition of binding of the antibody or the antigenbinding fragment thereof according to the present invention can bedetermined.

In general, inhibition of binding shows the presence of anti-ZIKV NS1antibodies in the sample of the subject, thus indicating ZIKV infectionof the subject. In samples of non-infected subjects, in contrast,typically no inhibition of binding is expected. Importantly, such anassay using the ZIKV NS1-binding antibodies of the present inventiondoes not score positive in subjects that were already infected withother flaviviruses. Flaviviruses typically induce a large number ofantibodies that are cross-reactive with ZIKV. In other words this assayis highly specific and not affected by cross-reactive Abs.

Accordingly, the present invention also provides a Blockade-of-bindingassay for in-vitro diagnosis of Zika virus infection comprising thefollowing steps:

-   -   (i) adding an isolated sample from a subject to be diagnosed to        a plate coated with ZIKV NS1 protein and incubating said sample        on said plate,    -   (ii) adding an antibody, or an antigen binding fragment thereof,        that specifically binds to NS1 protein of ZIKA virus or a        complex including the antibody,    -   (iii) determining inhibition of binding of said antibody or        antigen-binding fragment thereof.    -   Preferably, the isolated sample from a subject to be diagnosed        is selected from blood, saliva and urine; preferably the sample        is a blood sample, such as whole blood, plasma or serum.

It is also preferred that the antibody, or the antigen-binding fragmentthereof, added in step (ii), is labelled, preferably biotinylated orconjugated to horseradish peroxidase (HRP).

Moreover, the isolated sample from a subject to be diagnosed ispreferably diluted, for example 1:5-1:50, preferably 1:5-1:25, such as1:10.

Preferably, the incubation time in step (i) is at least 5 min,preferably at least 15 min, more preferably at least 30 min, even morepreferably at least 45 min and most preferably at least 60 min.

It is furthermore preferred that in step (ii) after adding the antibody,or the antigen binding fragment thereof, the antibody, or the antigenbinding fragment thereof, is incubated for at least 1 min, preferably,at least 3 min, more preferably at least 5 min, even more preferably atleast 10 min and most preferably at least 15 min.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention, which is used as probe antibody inthe blockade of binding assay is a preferred antibody, or antigenbinding fragment thereof, according to the present invention. Forexample, the antibody, or the antigen binding fragment thereof,according to the present invention may preferably be an antibody, orantigen binding fragment thereof, according to the present inventionbinding to antigenic site S2 of Zika virus NS1 protein. Most preferably,the antibody, or the antigen binding fragment thereof, according to thepresent invention, which is used as probe antibody in the blockade ofbinding assay is an antibody, or the antigen binding fragment thereof,comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1,CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs:127-131 and 133; or functional sequence variants thereof having at least70%, at least 75%, at least 80%, at least 85%, at least 88%, at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs:127-130 and 132-133; or functional sequence variants thereof having atleast 70%, at least 75%, at least 80%, at least 85%, at least 88%, atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity. Particularly preferably,the antibody, or the antigen binding fragment thereof, according to thepresent invention, which is used as probe antibody in the blockade ofbinding assay is an antibody, or the antigen binding fragment thereof,comprising a heavy chain variable region (VH) amino acid sequenceaccording to SEQ ID NO: 134 or a functional sequence variant thereofhaving at least 70%, at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity and/or a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 135 ora functional sequence variant thereof having at least 70%, at least 75%,at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity.

For example, inhibition of binding of exemplary biotinylated antibodiesor antigen binding fragments thereof according to the present inventionmay be assessed by determining the optimal concentration of the antibodyor the antigen binding fragment thereof according to the presentinvention to achieve 70% maximal binding to ZIKV NS1 protein. Forexample, the optimal concentrations of the exemplary antibodies gZKA15,gZKA25 and gZKA35 to achieve 70% maximal binding to ZIKV NS1 protein maybe 38, 17 and 7 ng/ml, respectively. After performing the abovedescribed blockade-of-binding assay, substrate, such as p-NPP may beadded and the ELISA plate may be read at 405 nm and the percentage ofinhibition of binding may be calculated by the following equation (I):

% inhib=(1−

(OD sample−OD neg ctr)/(OD pos ctr−OD neg ctr)

)×100  (I)

wherein “% inhib” refers to the percentage of inhibition of binding ofthe antibody or the antigen binding fragment thereof according to thepresent invention to ZIKV NS1 protein; “OD sample” refers to the opticaldensity of the sample; “OD neg ctr” refers to the optical density of anegative control; and “OD pos ctr” refers to the optical density of apositive control.

This assay provides several advantages, such as the ability to detectclinical, sub-clinical and asymptomatic ZIKV infections at thepopulation level, being able to distinguish them from other flavivirusinfections, such as DENV. In particular, the diagnosis assay accordingto the present invention provides higher precision than direct ELISAbinding assays.

Moreover, the present invention also provides a method for (in vitro)diagnosis of Zika infection (in an isolated sample), wherein theantibody, or the antigen binding fragment thereof, according to thepresent invention, that binds to ZIKV NS1 protein, the complex accordingto the present invention, the composition according to the presentinvention or the kit of parts according to the present invention is usedfor determining whether an isolated sample (of a body fluid), such as anisolated blood sample, is infected with Zika virus.

Preferred embodiments of the above use in diagnosis apply also for thediagnosis method. For example, preferred isolated samples (of thesubject) include samples of a body fluid and tissue samples. A sample ofa body fluid is more preferred. Preferred body fluids for diagnosis ofZIKV infection include blood (e.g. whole blood, plasma, serum), salivaand urine. Blood, in particular plasma or serum, is most preferred.Moreover, preferred diagnosis assays are immunoassays. Preferredexamples of immunoassays include ELISA, immunofluorescence,immunohistochemistry and flow cytometry. Preferably, diagnosis includesELISA. Most preferably, a blockade-of-binding assay as described aboveis used.

Preferably, the method for (in vitro) diagnosis of Zika infection (in anisolated sample) comprises a step of

-   (i) contacting the isolated sample with the antibody, or the antigen    binding fragment thereof, according to the present invention, that    binds to ZIKV NS1 protein, the complex according to the present    invention, or the composition according to the present invention.

More preferably, the method for (in vitro) diagnosis of Zika infection(in an isolated sample) comprises the following steps:

-   (0) adding an isolated sample from a subject to be diagnosed (e.g.,    a sample of a body fluid, such as blood (e.g. whole blood, plasma,    serum), saliva and urine) to an ELISA plate coated with ZIKV NS1    protein;-   (i′) further adding the antibody or the antigen binding fragment    thereof according to the present invention to the ELISA plate,    wherein the antibody or the antigen binding fragment thereof    according to the present invention is preferably labelled, e.g.    biotinylated;-   (ii) optionally, washing the ELISA plate; and-   (iii) determining the inhibition of binding of the antibody or the    antigen binding fragment thereof according to the present invention.

In a further aspect, the present invention also provides theneutralizing antibody, or the antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention, or the pharmaceuticalcomposition according to the present invention for use according intreatment or prevention of ZIKV infection in subjects diagnosed withZika virus infection by using the antibody, or the antigen bindingfragment thereof, according to the present invention, that binds to ZIKVNS1 protein, the complex according to the present invention, thecomposition according to the present invention, the kit of partsaccording to the present invention or the method for ZIKV diagnosisaccording to the present invention.

In a further aspect, the present invention also provides a method ofpreventing and/or treating Zika virus infection, the method comprisingthe following steps:

-   (i) diagnosing Zika virus infection in a subject by using the    antibody, or the antigen binding fragment thereof, according to the    present invention, that binds to ZIKV NS1 protein, the complex    according to the present invention, the composition according to the    present invention, the kit of parts according to the present    invention or the method for ZIKV diagnosis according to the present    invention; and-   (ii) administering to said subject the neutralizing antibody, or the    antigen binding fragment thereof, according to the present    invention, the nucleic acid according to the present invention, the    vector according to the present invention, the cell according to the    present invention, or the pharmaceutical composition according to    the present invention.

Preferably, in this method of preventing and/or treating Zika virusinfection, step (i) of diagnosing Zika virus infection is performed asin-vitro diagnosis on an isolated sample (of a body fluid), such as anisolated blood sample.

In a further aspect, the present invention also provides a kit of partscomprising

-   (i) the antibody, or the antigen binding fragment thereof, according    to the present invention, that binds to ZIKV NS1 protein, the    complex according to the present invention, the composition    according to the present invention, or the kit of parts according to    the present invention; and-   (ii) the neutralizing antibody, or the antigen binding fragment    thereof, according to the present invention, the nucleic acid    according to the present invention, the vector according to the    present invention, the cell according to the present invention, or    the pharmaceutical composition according to the present invention.

Such a kit of parts is particularly useful in a method as describedabove. By use of such a method and/or such a kit, ZIKV infection can bespecifically diagnosed as well as prevented and/or treated.

DESCRIPTION OF FIGURES

FIG. 1 shows the reactivity (ELISA) and ZIKV and DENV1 neutralizingactivity of antibodies derived from four ZIKV immune donors (ZKA, ZKB,ZKC and ZKD) to E protein of ZIKV and DENV1-4 and to EDIII—domain ofZIKV E protein; NNB—neutralizing, non-E-protein binding antibodies.

FIG. 2 shows the reactivity (ELISA) of antibodies derived from four ZIKVimmune donors (ZKA, ZKB, ZKC and ZKD) to NS1 protein of ZIKV, DENV1-4and other flaviviruses. YFV—yellow-fever virus; WVN—West-Nile virus;JEV—Japanese Encephalitis virus; and TBEV—Tick-borne Encephalitis virus(nd, not determined).

FIG. 3 shows the binding of ZKA190, ZKA78 and ZKA64 antibodies to ZIKVand DENV1 E and to ZIKV EDIII proteins as measured by ELISA.

FIG. 4 shows the binding of ZKA185 and ZKA190 antibodies to ZIKV E,DENV1 VLP and to ZIKV EDIII proteins as measured by ELISA.

FIG. 5 shows the binding of ZKA15, ZKA25 and ZKA35 antibodies to ZIKVand DENV1-4 NS1 proteins as measured by ELISA.

FIG. 6 shows for Example 3 ZIKV NS1 protein antigenic site mapping usingcross-competition Octet-binding studies. (A-B) Cross-competition matrixperformed by Octet on 24 mAbs specific for ZIKV NS1 (A) orcross-reactive to DENV NS1 (B). +, lack of binding of the secondary Ab;+/−, partial loss of binding of the secondary mAb; binding of thesecondary mAb. Strikethrough cells, not tested. (C) Map of the antigenicsites targeted by ZIKV NS1-specific mAbs as defined using BLI (Octet)cross-competition.

FIG. 7 shows for Example 4 blockade of binding assay using mAb ZKA35 asa probe to detect ZIKV NS1 in plasma from ZIKV-immune (n=4), DENV-immune(n=5) and control donors (n=48) (1/10 dilution). Plasma samples weretested for their capacity to bind NS1 (empty dots) and to inhibit thebinding of the biotinylated

FIG. 8 shows for Example 5 the neutralizing activity of ZKA190, ZKA64,ZKA64-LALA, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain)and DENV1 on Vero cells as measured by flow-cytometry (% of infectedcells).

FIG. 9 shows for Example 5 the neutralizing activity of ZKA190, ZKA64,ZKA185, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain) onVero cells as measured with a cell viability readout (wst-1, Roche).

FIG. 10 shows for Example 6 the infection enhancing activity (ADE,antibody-dependent enhancement) of ZKA190, ZKA64, ZKA64-LALA, ZKA185,ZKA230 and ZKA78 antibodies for ZIKV (H/PF/2013 strain) onnon-permissive K562 cells as measured by flow-cytometry (% of infectedcells).

FIG. 11 shows for Example 6 that four ZIKV-immune plasma and oneDENV-immune plasma showed similar capacity to enhance ZIKV infection ofK562 cells (upper panel). This ADE effect was completely blocked in allfive immune plasma by the EDIII-specific ZKA64-LALA antibody (lowerpanel).

FIG. 12 shows the amino acid alignment of the EDIII region of 39 ZIKVstrains from the Asian lineage since 2013 (including the prototypicstrain MR766 of the African lineage isolated in 1947). The EDIII regionamino acid sequence of ZIK V H/PF/2013 (SEQ ID NO: 402) is shown at thetop of the alignment. Depicted EDIII sequences differing from ZIKVH/PF/2013 are those of strains MR766 (SEQ ID NO: 409), KU870645|USA|2Feb. 2016 (SEQ ID NO: 410), KU729218|Brazil|2015 (SEQ ID NO: 411),KU681081|Thailand|9 Jul. 2014 (SEQ ID NO: 412), KU926310|Brazil|29 Jan.2016 (SEQ ID NO: 413), and KU744693|China|6 Feb. 2016 (SEQ ID NO: 414).

FIG. 13 shows for Example 5 the neutralizing activity of ZKA190 andZKA190-LALA antibody against three strains of ZIKV (H/PF/2013, MR766 andMRS_OPY_Martinique_PaRi_2015) on Vero cells as measured byflow-cytometry (% of infected cells).

FIG. 14 shows for Example 7 NS1 blockade-of-binding analysis of Europeanresidents. Shown are the BOB values for samples collected in Italy andSwitzerland. Plotted are the BOB values in samples from ZIKV, primaryand secondary DENV-, WNV-, and CHIKV-infected individuals and a panel ofsamples from healthy blood donors from Switzerland.

FIG. 15 shows for Example 8 neutralization of ZKA190 and C8 mAbs testedagainst a panel of four strains of ZIKV, as determined by the percentageof infected Vero cells in the presence of increasing amounts of the mAbs(A). Shown are also the IC50 values (B) and statistics (C). Data arerepresentative of at least two independent experiments.

FIG. 16 shows for Example 9 the neutralization and enhancement of ZIKVinfection by antibody ZKA190. (A) Neutralization of ZIKV PRVABC59 straininfection of hNPCs by ZKA190, ZKA190-LALA and a control mAb asdetermined by plaque assay on Vero cells (left panel) and indirectimmunofluorescence of infected hNPCs using fluorophore-labelled anti-Eantibody (right panel). (B) ADE of ZIKV infection of non-permissive K562cells by ZKA190 and ZKA190-LALA. (C) ADE induced in K562 cells when ZIKVis pre-incubated with serial dilutions of plasma serum from differentZIKV-positive patients (left panel). When ZKA190 LALA is added to theZIKV-serum complexes, ADE is inhibited (right panel). (D) ADE induced inK562 cells when ZIKV is pre-incubated with serial dilutions of a prMcross-reactive mAb (DV62) derived from a DENV-immune donor. ZKA190-LALAinhibits ADE of ZIKV when complexed with prM-reactive antibody DV62. (E)Effect on ADE induced by peak enhancing dilution of a DENV2 plasma (leftpanel) or anti-prM DV62 mAb (right panel) by serial dilutions ofindicated mAbs.

FIG. 17 shows for Example 10 the identification of ZKA190 epitope andanalysis of its conservation in ZIKV strains. (A) Overlay of[¹⁵N,¹H]-HSQC spectra of ¹⁵N-labeled ZIKV EDIII in absence (black) orpresence (red) of unlabelled ZKA190 Fab. Differences identify EDIIIresidues affected by antibody binding. (B) NMR epitope mapping of ZKA190Fab in complex with ZKV EDIII. The chemical shift perturbation (CSP,y-axis) is plotted against the EDIII residue number. Residues affectedby antibody binding are in red. (C) Residues in FG loop identified byNMR epitope mapping is partially hidden in E protein mol A but largelyexposed in mols B and C. EDIII of E protein was coloured in blue.Residues identified by NMR epitope mapping are coloured in magentaexcept those in the FG loop are coloured in green. Adjacent E proteinsare shown as grey surface. (D) Level of amino acid residue conservationin ZKA190 epitope as calculated by the analysis of sequences from 217ZIKV strains found in ZIKV Resources (NCBI) databases as of Nov. 24,2016. (E) Open-book representation showing charge complementaritybetween the epitope and paratope of the docking result. Boundaries ofthe epitope and paratope are circled in green. The borders between heavyand light chains of Fab and its corresponding footprint on EDIII areshown as yellow dashed lines.

FIG. 18 shows for Example 10 the ZKA190 epitope identified by NMR andDocking. (A) Cartoon representation of the 12 lowest energy NMRstructures of ZIKV EDIII, with residues affected by ZKA190 binding inred. Flexibility in the N-terminus of the construct is apparent. (B)Model of the ZKA190:EDIII complex derived by computational docking andmolecular simulation validated by NMR results. The NMR identifiedepitope on EDIII (grey) is in red. The ZKA190 heavy and light chain arecolored in dark and light green, respectively. EDIII residues thataffect or not antibody binding when mutated are shown as orange and bluesticks, respectively. (C) NMR identified ZKA190 epitope (red) isaccessible on the virus surface (white).

FIG. 19 shows for Example 10 the binding of wt or mutated EDIII toZKA190 IgG. SPR data and binding kinetics are shown. EDIII mutants thataffect (red highlights) or do not affect binding are shown as indicatedin the figure.

FIG. 20 shows for Example if the results of the confocal microscopyexperiments. ZIKV incubated with a concentration exceeding 10'000-foldthe IC50 value of either ZKA190 Fab or full IgG were added to Verocells. The ZIKV:antibody complex is detected inside the cells (green)and co-localizes with endosomes (red, yellow overlay). Endosomes andacidic organelles are marked by Lysotracker red; Alexa-488 conjugatedZKA190 is in green. Nuclei are stained with DAPI (blue).

FIG. 21 shows for Example 12 prophylactic and therapeutic efficacy ofZKA190. (A) ZKA190 is strongly protective against ZIKV infection whenadministered prophylactically to mice (A129 in (A) and AG129 in (B))challenged with a lethal dose of ZIKV strain MP174.51. Experiments usedN=4-8 mice per group. Kaplan-Meier survival curves are shown (A).Significance was determined by using the Mantel-Cox log-rank test. PanelA, top left: ZKA190 at 5, 1 and 0.2 mg/kg versus Ctr mAb, P=0.0031;ZKA190 at 0.04 mg/kg versus Ctr mAb, P=0.0116; ZKA190-LALA at 5, 1, 0.2and 0.04 mg/kg versus Ctr mAb, P=0.0031. Panel A, top right: Morbidityscore of mice monitored over a 14-15 day period (two different scoringmethods were used; see (Dowall, S. D., Graham, V. A., Rayner, E.,Atkinson, B., Hall, G., Watson, R. J., Bosworth, A., Bonney, L. C.,Kitchen, S., and Hewson, R. (2016). A Susceptible Mouse Model for ZikaVirus Infection. PLoS Negl Trop Dis 10, e0004658-13). Panel A, lowerpanels: body weight of mice. Panels B: ZKA190 or ZKA190-LALA wereadministered at 15 mg/kg at different time-points after ZIKV infection.Panel B, top left: A Kaplan-Meier survival curve is shown. Experimentsused N=5 mice per group. Significance was determined by using theMantel-Cox log-rank test. ZKA190 and ZKA190-LALA given either on day 1,2, 3 or 4 versus Ctr., P=0.0016. Panel B, top right: Morbidity score ofmice monitored over a 14-day according to (Dowall et al., 2016). Micewere monitored over a 14 day period for body weight loss (Panel B, lowerpanels). Control antibody is MPE8 specific for RSV F protein (Corti, D.,et al. Cross-neutralization of four paramyxoviruses by a humanmonoclonal antibody. Nature 501, 439-443 (2013)).

FIG. 22 shows for Example 12 the prophylactic efficacy of the anti-ZIKVEDIII-specific mAb ZKA190 against ZIKV strains MP1741. (A) Shown is theviremia measured as PFU/ml on day 5 in blood of all animals. (B) Viralload was measured as genomic copies/ml by qPCR on day 5 in blood of allanimals and in blood and indicated tissues when animals were culled atthe end of the study or when the humane end points were met. (C) Micewere monitored over a 14 day period for body weight loss (D) Human serumIgG concentration in day 5 blood samples. Significance was determinedcompared to control antibody treatment by nonparametric unpairedMann-Whitney U test. *p<0.05; **p<0.01; ***p<0.001.

FIG. 23 shows for Example 12 the therapeutic efficacy of the anti-ZIKVEDIII-specific mAb ZKA190. (A) Viral loads were measured as PFUs on day5 in blood of all animals. (B) Viral loads were measured as genomiccopies by qPCR on day 5 in blood of all animals and in blood andindicated tissues when animals were culled at the end of the study orwhen the human end points were met. Significance was determined comparedto control antibody treatment by nonparametric unpaired Mann-Whitney Utest. *p<0.05; **p<0.01. (C) Human serum IgG concentration in day 5blood samples.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1: Isolation of ZIKV-Specific Antibodies and Production ofMonoclonal Antibodies

IgG+ memory B cells were isolated from cryopreserved peripheral bloodmononuclear cells (PBMCs) of four ZIKV-infected donors (ZKA, ZKB, ZKCand ZKD) using CD22 microbeads (Miltenyi Biotec), followed by depletionof cells carrying IgM, IgD and IgA by cell sorting. Memory B cells fromthe ZIKV-infected donors were then immortalized with EBV (Epstein BarrVirus) and CpG (CpG oligodeoxynucleotide 2006) in multiple replicatewells as previously described (Traggiai, E. et al., Nat. Med. 10,871-875, 2004) and culture supernatants were then tested in a primaryscreening using in parallel a 384-well based micro-neutralization assayand a binding assay (ELISA) to test their binding to ZIKV NS1 protein orto ZIKV E protein. Results of the binding assay are shown in FIG. 1(binding to ZIKV E protein) and FIG. 2 (binding to ZIKV NS1 protein).

Neutralization assays were undertaken on Vero cells. In a 384-wellplate, ZIKV H/PF/2013 that resulted in an infection rate (m.o.i,multiplicity of infection) of 0.35 was incubated with superntanants for1 h at 37% (5% CO2) before the addition to pre-seeded 5'000 Vero cells.These were incubated for a further 5 days, after which supernatant wasremoved and WST-1 reagent (Roche) was added. Positive cultures werecollected and expanded. From positive cultures the VH and VL sequenceswere retrieved by RT-PCR. Antibodies were cloned into human IgG1 and Igkappa or Ig lambda expression vectors (kindly provided by MichelNussenzweig, Rockefeller University, New York, US) essentially asdescribed (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig M C,Wardemann H (2008) Efficient generation of monoclonal antibodies fromsingle human B cells by single cell RT-PCR and expression vectorcloning. J Immunol Methods 329: 112-124). Monoclonal antibodies wereproduced from EBV-immortalized B cells or by transient transfection of293 Freestyle cells (Invitrogen). Supernatants from B cells ortransfected cells were collected and IgG were affinity purified byProtein A or Protein G chromatography (GE Healthcare) and desaltedagainst PBS.

FIG. 1 provides an overview over selected ZIKV neutralizing antibodies(cf. Tables 1 and 2 for the amino acid sequences of their CDRs andheavy/light chain variable regions). The last two columns of FIG. 1provide the neutralization activities (IC₅₀) of ZIKV and DENV1 (iftested). The other columns provide binding activities (EC₅₀) of theantibodies to ZIKV E protein (ZIKV E), DENV1 E protein (DENV1 E), DENV2E protein (DENV2 E), DENV3 E protein (DENV3 E), DENV4 E protein (DENV4E), DENV1 virus-like particle (DENV1 VLP), DENV2 virus-like particle(DENV2 VLP), DENV3 virus-like particle (DENV3 VLP), DENV4 virus-likeparticle (DENV4 VLP), and to EDIII-domain of ZIKV E protein (DIII ZKA).

Additional antibodies were isolated for their ability to bind to ZIKVNS1 protein (cf. FIG. 2). Positive cultures were collected and expanded.From positive cultures the VH and VL sequences were retrieved by RT-PCR.Antibodies were cloned into human IgG1 and Ig kappa or Ig lambdaexpression vectors (kindly provided by Michel Nussenzweig, RockefellerUniversity, New York, US) essentially as described (Tiller T, Meffre E,Yurasov S, Tsuiji M, Nussenzweig M C, Wardemann H (2008) Efficientgeneration of monoclonal antibodies from single human B cells by singlecell RT-PCR and expression vector cloning. J Immunol Methods 329:112-124). Monoclonal antibodies were produced from EBV-immortalized Bcells or by transient transfection of 293 Freestyle cells (Invitrogen).Supernatants from B cells or transfected cells were collected and IgGwere affinity purified by Protein A or Protein G chromatography (GEHealthcare) and desalted against PBS.

FIG. 2 provides an overview over selected ZIKV NS1-protein bindingantibodies (cf. Tables 4 and 5 for the amino acid sequences of theirCDRs and heavy/light chain variable regions). Namely, FIG. 2 providesbinding activities (EC₅₀) of the antibodies to ZIKV NS1 protein (ZIKVNS1), DENV1 NS1 protein (DENV1 NS1), DENV2 NS1 protein (DENV2 NS1),DENV3 NS1 protein (DENV3 NS1), DENV4 NS1 protein (DENV4 NS1),yellow-fever virus NS1 protein (YFV NS1), West-Nile virus NS1 protein(WNV NS1), Japanese-Encephalitis virus NS1 protein (JEV NS1), and toTick-borne Encephalitis virus NS1 protein (TBEV NS1).

Example 2: Characterization of Antibodies ZKA190, ZKA185, ZKA230, ZKA64and ZKA78

In Example 1, a large number of ZIKV-neutralizing antibodies wereidentified and characterized for their specificity to ZIKV, inparticular ZIKV E protein and ZIKV EDIII as well as for theircross-reactivity towards DENV. Antibodies ZKA190 (SEQ ID NOs: 1-18),ZKA185 (SEQ ID NOs: 19-36), ZKA230 (SEQ ID NOs: 37-54), ZKA64 (SEQ IDNOs: 73-90) and ZKA 78 (SEQ ID NOs: 55-72) described in Example 1 werethen selected and further tested against ZIKV E protein (“ZIKV”), ZIKVEDIII (“DIIIZI”) and also tested against the E protein of dengue virus(DENV, serotype number 1) by ELISA. To this end, a standard ELISA wasused. Briefly, ELISA plates were coated with ZIKV E protein at 1 or 3μg/ml, blocked with 10% FCS in PBS, incubated with sera or humanantibodies and washed. Bound antibodies were detected by incubation withAP-conjugated goat anti-human IgG (Southern Biotech). Plates were thenwashed, substrate (p-NPP, Sigma) was added and plates were read at 405nm. The relative affinities of monoclonal antibody binding weredetermined by measuring the concentration of antibody (EC50) required toachieve 50% maximal binding at saturation.

Results are shown in FIGS. 3 and 4. Of note, ZKA64 and ZKA190 bound toZIKV E and ZIKV EDIII (“DIII ZI”) with low EC50 values, therebyindicating that ZKA64 and ZKA190 are binding to domain III of ZIKV Eprotein (EDIII). ZKA78 bound to ZIKV E, but not to ZIKV EDIII,indicating that ZKA78 is binding to ZIKV E, but not targeting the EDIIIregion. Despite their considerable ZIKV neutralizing activity (cf. FIG.1), antibodies ZKA185 and ZKA230 did not show any detectable binding toZIKV E and ZIKV EDIII (FIG. 4). Accordingly, ZKA185 and ZKA230 werereferred to as “neutralizing-non-E-binding” (NNB) antibodies. Those NNBantibodies are assumed to recognize quaternary epitopes that aredisplayed on the ZIKV infectious virions but not on soluble proteins.

Moreover, none of ZKA190, ZKA185, ZKA230, and ZKA64 showed anydetectable binding to DENV E proteins (FIG. 1, DENV1-4 serotypes, andFIGS. 3 and 4), indicating that ZKA190, ZKA185, ZKA230, and ZKA64 arespecific for ZIKV and not cross-reactive to dengue virus. ZKA78, incontrast, which is assumed to bind to ZIKV EDI/II, but not to ZIKV EDIII(cf. FIG. 3), bound to DENV E proteins (FIGS. 1 and 3), indicating thatZKA78 is a cross-reactive antibody binding to both, ZIKV and DENV.

To further confirm those results, the ZIKV E protein binding antibodiesZKA190, ZKA64 and ZKA78 were additionally tested against E protein ofdengue virus (DENV, serotypes number 1-4). ZKA64 and ZKA190 did not bindto DENV1-4 E protein, thereby confirming that ZKA64 and ZKA190 arespecific for ZIKV. ZKA78, in contrast, bound to DENV1-4 E, confirmingthat ZKA78 is a cross-reactive antibody binding to the E protein of bothZIKV and DENV (cf. FIG. 1).

Example 3: Characterization of ZIKV NS1-Specific Antibodies forSerological Diagnosis

In Example 1, a large number of NS1-reactive antibodies were identifiedand then characterized for their specificity to ZIKV NS1 andcross-reactivity towards other flavivirus NS1 proteins (FIG. 2).Antibodies ZKA15 (SEQ ID NOs: 91-108), ZKA25 (SEQ ID NOs: 109-126) andZKA35 (SEQ ID NOs: 127-144) were then further characterized for bindingto ZIKV NS1 and DENV1 NS1, DENV2 NS1, DENV3 NS1 and DENV4 NS1. To thisend, a standard ELISA was used. Briefly, ELISA plates were coated withZIKV NS1 protein at 1 μg/ml, blocked with 10% FCS in PBS, incubated withsera or human antibodies and washed. Bound antibodies were detected byincubation with AP-conjugated goat anti-human IgG (Southern Biotech).Plates were then washed, substrate (p-NPP, Sigma) was added and plateswere read at 405 nm. The relative affinities of monoclonal antibodybinding were determined by measuring the concentration of antibody(EC50) required to achieve 50% maximal binding at saturation.

Results are shown in FIG. 5. All three antibodies (ZKA15, ZKA25 andZKA35) bound with high affinity to ZIKV NS1 but not to the DENV1-4 NS1antigens (FIG. 5).

To investigate the binding of the antibodies to ZIKV NS1 further,bio-layer interferometry competition assays were used. Across-competition matrix was generated using biolayer interferometry(BLI; Octet) on 13 antibodies specific for ZIKV NS1 (i.e. notcross-reactive with DENV NS1), namely antibodies ZKA24, ZKA15, ZKA32,ZKA19, ZKA50, ZKA37, ZKA46, ZKA10, ZKA48, ZKA35, ZKA25, ZKA44, and ZKA30(cf. FIG. 6A). As can be retrieved from FIG. 2 none of those 13antibodies showed detectable binding to DENV NS1.

Competition assays and antigenic sites determination were determined at37° C. with a Octet RED96 system, ForteBio. The ZIKV-NS1 protein dilutedto 2.5 μg/ml in PBS was immobilized for 7-9 minutes on the surface of anAPS coated sensor-chip. Coated biosensors were placed in wellscontaining blocking buffer (0.1% BSA in PBS) for 6 minutes to block freeBiosensor binding sites. Coated-Biosensors were then incubated for 8minutes with a set of single purified mAbs specific for ZIKV-NS1 dilutedin blocking buffer at 10 μg/ml. After binding of the first set of mAbs(step 1), Biosensors were moved to wells containing different mAbs for 8minutes (step 2). Association of the second mAb resulted in recognitionof a different antigenic site compared to the first mAb (e.g.non-competition). Competition or partial competition were determined instep 2 when no association or low association was detected,respectively. A cross-competition matrix was created by multiple runs ofcompetitions in order to predict antigenic site mapping on ZIKV NS1.

Results are shown in FIGS. 6A and 6C. Firstly, all of the ZIKVNS1-specific antibodies tested were binding to antigenic site(s) S1and/or S2 (FIG. 6A). However, some of the antibodies did not competewith others. For example, ZKA15 did not compete for binding with ZKA25and ZKA35 and vice versa (FIG. 6A). Accordingly, antibody ZKA15 wasassigned to the antigenic site S1, while antibodies ZKA25 and ZKA35 wereassigned to the antigenic site S2 (FIG. 6C). In summary, based on theantibodies used, antigenic sites (S1 and S2) on ZIKV NS1 were identified(FIG. 6C).

Additionally, binding of 10 antibodies cross-reacting to ZIKV NS1protein and to DENV NS protein (namely, ZKA18, ZKA29, ZKA39, ZKA53,ZKA54, ZKB19, ZKB23, ZKC29, ZKC33, and ZKC34; FIG. 6B) to antigenicsites S1 and/or S2 on ZIKV NS1 was investigated. As can be retrievedfrom FIG. 2 all of those 10 antibodies showed binding to DENV NS1. Those10 cross-reactive antibodies were tested in a cross-competition assay asdescribed above (for the ZIKV NS1-specific antibodies) against ZIKV NS1S1-specific antibody ZKA15 and against ZIKV NS1 S2-specific antibodyZKA35.

Results are shown in FIG. 6B. Interestingly, none of the tencross-reactive antibodies tested competed with ZKA 15 and/or ZKA35 forbinding to antigenic site(s) S1 and/or S2 on ZIKV NS (FIG. 6B). Theseresults show that ZKA15 and ZKA35 antigenic site is not targeted by NScross-reactive antibodies. Thus, NS1 antigenic sites S1 and S2 weretargeted by ZIKV-specific, but not by cross-reactive antibodies.

Example 4: Use of ZIKV NS1-Specific Antibodies in Diagnosis of ZIKVInfection

In the present Example, the usefulness of the ZIKV NS1-specificantibodies of the present invention in diagnosis of ZIKV infection wasinvestigated. More specifically, the use of ZIKV NS1-specific antibodiesof the present invention to specifically detect the presence or absenceof antibodies elicited against ZIKV NS1 in plasma samples of ZIKV- orDENV-infected donors was determined.

To this end, a “blockade of binding” assay was used. In particular, theability of ZIKV NS1-reactive plasma antibodies to inhibit the binding ofthe biotinylated antibody ZKA35 to ZIKV NS1 was measured. To this end,ZIKV NS1-specific antibody ZKA35 was biotinylated using the EZ-LinkNHS-PEO solid phase biotinylation kit (Pierce). Labelled ZKA35 wastested for binding to ZIKV NS1 to determine the optimal concentration ofZKA35 to achieve 70% maximal binding. Plasma samples from ZIKV- (n=4),DENV-immune (n=5) donors and control (n=48) plasma (1/10 dilution) wereadded to ELISA plates coated with ZIKV NS1. After 1 h, biotinylatedanti-ZIKV NS1 antibody ZKA35 was added at the concentration achieving70% maximal binding and the mixture was incubated at room temperaturefor 15 minutes. Plates were washed, substrate (p-NPP, Sigma) was addedand plates were read at 405 nm. The percentage of inhibition wascalculated as follow: (1−

(OD sample−OD neg ctr)/(OD pos ctr−OD neg ctr)

)×100.

Results are shown in FIG. 7. Of note, antibody ZKA35 binding to theantigenic site S2 on NS was inhibited only by plasma samples fromZIKV-immune donors, but not DENV-immune donors, and its binding was alsonot inhibited by 48 control plasma samples (FIG. 7). Accordingly, thisassay may be used as to specifically detect clinical and sub-clinicalZIKV infections at the population level.

Example 5: The Antibodies According to the Present Invention PotentlyNeutralize ZIKV Infection

The isolated antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 weretested for their ability to neutralize ZIKV and DENV1 infection invitro.

Neutralization of DENV and ZIKV infection by antibodies was measuredusing a micro-neutralization flow cytometry-based assay. Differentdilutions of antibodies were mixed with ZIKV (MOI of 0.35) or attenuatedDENV1 (all at MOI of 0.04) for 1 hour at 37° C. and added to 5000 Verocells/well in 96-well flat-bottom plates. After four days for ZIKV andfive days for DENV, the cells were fixed with 2% formaldehyde,permeabilized in PBS 1% FCS 0.5% saponin, and stained with the mouse mAb4G2. The cells were incubated with a goat anti-mouse IgG conjugated toAlexa Fluor488 (Jackson Immuno-Research, 115485164) and analyzed by flowcytometry. In other cases the ZIKV neutralization data are alsodetermined measuring cell viability using the WST-1 reagent (Roche). Theneutralization titer (50% inhibitory concentration

IC50

) was expressed as the antibody concentration that reduced the infectionby 50% compared to cell-only control wells.

Results are shown in FIGS. 8, 9 and 13. The EDIII-specific mAbs ZKA64and ZKA190 and the NNB mAb ZKA230 were highly potent in ZIKVneutralization (strain H/PF/2013), with IC50 values of 93, 9 and 10ng/ml, respectively (FIG. 8, upper panel). In contrast, thecross-reactive antibody ZKA78 only partially neutralized ZIKVinfectivity and cross-neutralized DENV1 infectivity (FIG. 8, lowerpanels). Similar data were obtained by measuring the ZIKV-inducedcytopathic effect as measured with the WST-1 reagent (FIG. 9). In thissecond assay, NNB antibody ZKA185 was also included in the panel oftested antibodies and showed an IC50 similar to the most potentantibodies ZKA190 (EDIII-specific) and ZKA230 (NNB).

It is important to note that the ultra-potent ZKA64 and ZKA190antibodies in addition to their ability to neutralize the ZIKV H/PH/2013strain (present example), also bound to the E protein and EDIII derivedfrom the ZIKV strains MR766 and SPH2015, respectively (FIG. 1 and FIG.3). ZKA190 and ZKA190-LALA was also confirmed to effectively neutralizetwo additional ZIKV strains (MR766 and MRS_OPY_Martinique_PaRi_2015)(FIG. 13). Taken together the results indicate that the ultra-potentZKA64 and ZKA190 antibodies cross-react with multiple strains of ZIKVbelonging to different genotypes and origins (East African and Asianfrom Uganda, French Polynesia, Martinique and Brazil).

Example 6: The “LALA” Mutation Inhibits Antibody-Dependent Enhancementof ZIKV Infection by Serum Antibodies

Neutralizing antibodies were also tested for their ability to enhancethe infection of ZIKV in the non-permissive K562 cells(antibody-dependent enhancement assay, ADE assay). ADE was measured by aflow based assay using K562 cells. Antibodies and ZIKV H/PF/2013 (MOI0.175) were mixed for 1 hour at 37° C. and added to 5000 K562cells/well. After four days, cells were fixed, permeabilized, andstained with m4G2. The number of infected cells was determined by flowcytometry.

Results are shown in FIG. 10. All antibodies enhanced infection of ZIKVin the non-permissive K562 cells at a broad range of concentrations,including those that fully neutralized ZIKV infection on Vero cells(FIG. 10). Of note, while EDIII-specific antibodies ZKA64 and ZKA190fully neutralized ZIKV infections of K562 cells above 1 μg/ml, the NNBantibody ZKA230 failed to do so, a result that might be due to thedifferent mechanisms of neutralization of free viruses versusFc-gamma-receptor-internalized viruses. In contrast, the cross-reactiveZKA78 that only partially neutralized ZIKV infectivity, effectivelyenhanced ZIKV infection of K562 cells. These results show thatcross-reactive antibodies elicited by either ZIKV or DENV infection canmediate heterologous ADE.

In view thereof it was investigated whether ADE could be also induced byimmune sera and whether this could be blocked by neutralizing antibodiesdelivered as a “LALA variant”. To obtain the LALA variant, each of theheavy chains was mutated at amino acids 4 and 5 of CH2 domain bysubstituting an alanine in place of the natural leucine usingsite-directed mutagenesis. As described above, LALA variants (of humanIgG1 antibodies) do not bind to Fc-gamma-receptors and complement.

To investigate the effect of ZKA64-LALA antibody in ZIKV ADE, aninhibition of ADE assay was used. Since ADE of ZIKV is observed usingZIKV- or DENV-immune plasma, ZIKV (MOI 0.175) was mixed with plasma fromprimary ZIKV- or DENV-infected donors for 30 minutes at 37° C.ZKA64-LALA antibody was added at 50 μg/ml, mixed with 5000 K562cells/well and incubated for three days. Cells were then stained with4G2 and analyzed by flow cytometry.

Results are shown in FIG. 11. In a homologous setting, four ZIKV-immuneplasma collected from convalescent patients and one DENV-immune plasmashowed similar capacity to enhance ZIKV infection of K562 cells (FIG.11, upper panel), and this ADE effect was completely blocked by theEDIII-specific ZKA64-LALA antibody (FIG. 11, lower panel).

Of note, the ADE effect of ZIKV- and DENV-immune plasma was completelyblocked by the EDIII-specific ZKA64-LALA antibody. The ADE blockingability of a single EDIII-specific LALA antibody could be related notonly to its capacity to out-compete serum enhancing antibodies but alsoto neutralize virus once internalized into endosomes.

These results indicate that a potently neutralizing antibody, such asZKA190, ZKA230, ZKA185 or ZKA64, developed in the “LALA” form, have astrong potential to be used in prophylactic or therapeutic settings toprevent congenital ZIKV infection, e.g. in pregnant women and/or inpeople living in high risk areas. The use of the LALA form avoids therisk of ZIKV ADE and, as shown above, could also block ADE ofpre-existing cross-reactive antibodies, such as in the case of patientsalready immune to DENV.

Example 7: Analysis of Samples from European Residents Using ZIKVNS1-Specific Antibodies for Diagnosis of ZIKV Infection

The present Example is based on the blockade of binding assay describedin Example 4. To further assess the specificity of the ZIKV NS1 BOBassay, a large set of samples obtained from patients infected with DENV,WNV or Chikungunya virus (CHIKV) was tested.

To this end, a “blockade of binding” assay was used. Polystyrene plateswere coated overnight with 1 μg/ml of ZIKV NS1 and blocked for 1 hourwith PBS containing 1% BSA. Plasma or serum (1:10 dilution) were addedto NS1-coated ELISA plates. Thereafter, e.g. after 1 hour, an equalvolume of biotinylated anti-NS1 ZKA35 was added, and the mixture wasincubated, e.g. at room temperature for 15 minutes. Plates were washedand alkaline-phosphatase-conjugated streptavidin was added, e.g. for 30minutes. Plates were washed again and the substrate was added. Thepercentage of inhibition was calculated as follow: (1−

(OD sample−OD neg ctr)/(OD pos ctr−OD neg ctr)

)×100.

Results are shown in FIG. 14. Thirty-one of 32 samples (96.9%) from WNVpatients collected more than 10 days after symptom onset scorednegative. Of note, the only positive was obtained from a samplecollected in 2016. Two of 27 samples from DENV patients collected morethan 10 days after symptom onset scored positive, and the two positivesamples were derived from secondary DENV infections. In addition, noneof the samples derived from chikungunya patients or YFV-vaccines scoredpositive. A large number of plasma samples from Swiss blood donors(n=116) collected between 2010 and 2016 was also tested. None of thosesamples scored positive. The results obtained confirmed and strengthenedthe high sensitivity and specificity of the NS1 BOB ELISA assay.

Example 8: An Antibody According to the Present Invention NeutralizesZIKV More Potently than Prior Art Antibody EDE1 mAb C8

To compare the neutralizing antibodies according to the presentinvention with highly neutralizing anti-ZIKV antibodies of the priorart, neutralization performance of ZKA190 was compared to that of priorart highly neutralizing mAb EDE1 C8 (Barba-Spaeth G, Dejnirattisai W,Rouvinski A, Vaney M C, Medits I, Sharma A, Simon-Lorière E, SakuntabhaiA, Cao-Lormeau V M, Haouz A, England P, Stiasny K, Mongkolsapaya J,Heinz F X, Screaton G R, Rey F A. Structural basis of potent Zika-denguevirus antibody cross-neutralization. Nature. 2016 Aug. 4;536(7614):48-53). Neutralization of both antibodies was tested against apanel of four distinct ZIKV strains (H/PF/2013; MR766, MRS-OPY andPV10552).

Briefly, neutralization of ZIKV infection by mAbs was measured using amicro-neutralization flow cytometry-based assay. Different dilutions ofmAbs were mixed with ZIKV (MOI of 0.35) for 1 hour at 37° C. and addedto 5000 Vero cells/well in 96-well flat-bottom plates. After four daysfor ZIKV, the cells were fixed with 2% formaldehyde, permeabilized inPBS containing 1% fetal calf serum (Hyclone) and 0.5% saponin, andstained with the mouse mAb 4G2. The cells were incubated with a goatanti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research,115485164) and analyzed by flow cytometry. The neutralization titer (50%inhibitory concentration

IC50

) is expressed as the antibody concentration that reduced the infectionby 50% compared to virus-only control wells.

Results are shown in FIG. 15. ZKA190 mAb potently neutralized African,Asian and American strains with an IC50 ranging from 0.6 to 8 ng/ml. Incomparison, prior art antibody C8 was about 24-fold less potent.

Example 9: Further Characterization of Antibody ZKA190

The potency of antibody ZKA190 was further investigated in vitro and invivo. To this end, the mAb was synthesized in IgG1 wild-type (wt) formatand in an IgG1 Fc-LALA format. Briefly, the VH and VL sequences werecloned into human Igγ1, Igκ and Igλ expression vectors (kindly providedby Michel Nussenzweig, Rockefeller University, New York, N.Y., USA),essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M,Nussenzweig M C, Wardemann H: Efficient generation of monoclonalantibodies from single human B cells by single cell RT-PCR andexpression vector cloning. J Immunol Methods 2008, 329:112-124).Recombinant mAbs were produced by transient transfection of EXPI293cells (Invitrogen), purified by Protein A chromatography (GE Healthcare)and desalted against PBS. To obtain the LALA variant, each of the heavychains was mutated at amino acids 4 and 5 of CH2 domain by substitutingan alanine in place of the natural leucine using site-directedmutagenesis. As described above, LALA variants (of human IgG1antibodies) do not bind to Fc-gamma-receptors and complement.

As shown in FIG. 15A and described in Example 8, ZKA190 was testedagainst a panel of four ZIKV strains. ZKA190 mAb potently neutralizedAfrican, Asian and American strains with an IC50 ranging from 0.004 to0.05 nM (FIG. 15A; 0.6 to 8 ng/ml).

Since ZIKV has been shown to infect human neural progenitor cells (hNPC)leading to heightened cell toxicity, dysregulation of cell-cycle andreduced cell growth, ZKA190 and ZKA190-LALA were tested in hNPCs. Tothis end, adult male fibroblasts obtained from the Movement DisordersBio-Bank (Neurogenetics Unit of the Neurological Institute ‘CarloBesta’, Milan) were reprogrammed using the CytoTune-iPS 2.0 Sendai kit(Life Technologies). hiPSCs were maintained in feeder-free conditions inmTeSR1 (Stem Cell Technologies). To generate embryoid bodies (EBs),dissociated hiPSCs were plated into low adhesion plates in mTeSR1supplemented with N2 (0.5×) (ThermoFisher Scientific), human Noggin (0.5mg/ml, R&D System), SB431542 (5 M, Sigma), Y27632 (10 μM, MiltenyiBiotec) and penicillin/streptomycin (1%, Sigma) (as described inMarchetto M C N, Carromeu C, Acab A, Yu D, Yeo G W, Mu Y, Chen G, Gage FH, Muotri A R: A model for neural development and treatment of Rettsyndrome using human induced pluripotent stem cells. Cell 2010,143:527-539). To obtain rosettes, EBs were plated after 10 days ontomatrigel-coated plates (1:100, matrigel growth factor reduced, Corning)in DMEM/F12 (Sigma) with N2 (1:100), non-essential amino acids (1%,ThermoFisher Scientific) and penicillin/streptomycin. After 10 days,cells were passaged with Accutase (Sigma) and seeded onto matrigelcoated-flasks in NPC media containing DMEM/F12, N2 (0.25%), B27 (0.5%,ThermoFisher Scientific), penicillin/streptomycinand FGF2 (20 ng/ml,ThermoFisher Scientific). hNPCs (3×104) were plated on coverslips in24-well plates 3 days prior to infection with PRVABC59 strain. Virusstock was incubated with the mAbs 1 h prior to addition to hNPCs toobtain an MOI of 0.5. After 4 h of virus adsorption, culture supernatantwas removed and fresh medium containing the mAbs was re-added.Supernatant was collected 96 h post-infection to measure virus titers byplaque assay on Vero cells. Cells were fixed in 4% paraformaldehyde(PFA, Sigma) solution in phosphate-buffered saline (PBS, Euroclone) for30 min for indirect immunofluorescence. Fixed cells were permeabilizedfor 30 minutes (min) in blocking solution, containing 0.2% Triton X-100(Sigma) and 10% donkey serum (Sigma), and incubated overnight at 4° C.with the primary antibodies in blocking solution. The following antibodywas used for detection: anti-envelope (1:200, Millipore, MAB10216).Then, cells were washed with PBS and incubated for 1 h with Hoechst andanti-mouse Alexa Fluor-488 secondary antibodies (1:1,000 in blockingsolution, ThermoFisher Scientific). After PBS washes, cells were washedagain and mounted. Results are shown in FIG. 16A. Both, ZKA190 andZKA190-LALA, fully abolished infection and replication of ZIKV in hNPCs.

Next, the ability of ZKA190 and ZKA190-LALA to cause ADE was tested inthe K562 cell line as described in Example 6. Briefly, ADE was measuredby a flow based assay using K562 cells. Briefly, for ZKA190, ZKA190 andZIKV H/PF/2013 (MOI 0.175) were mixed for 1 hour at 37° C. and added to5000 K562 cells/well. After four days, cells were fixed, permeabilized,and stained with mAb m4G2. The number of infected cells was determinedby flow cytometry. For ZKA190-LALA, ZIKV (MOI 0.175) was mixed withplasma from primary ZIKV-infected donors for 30 minutes at 37° C.ZKA190-LALA was added at 50 μg/ml, mixed with 5000 K562 cells/well andincubated for three days. Cells were then stained with 4G2 and analyzedby flow cytometry. Results are shown in FIG. 16B. ZKA190 supports ADEfrom 0.0001 to 1 nM; as expected, ZKA190-LALA did not show any ADEactivity. The ability of ZKA190-LALA to inhibit ADE induced by plasmafrom four ZIKV-immune donors in K562 cells was also tested. Results areshown in FIG. 16C. It was found that ZKA190-LALA completely inhibitedthe ADE induced by plasma antibodies (FIG. 16C).

Anti-prM antibodies form part of the predominant antibodies elicitedduring the human immune response against flaviviruses and have beenshown to enhance virus infection in vitro (Dejnirattisai, W.,Jumnainsong, A., Onsirisakul, N., Fitton, P., Vasanawathana, S.,Limpitikul, W., Puttikhunt, C., Edwards, C., Duangchinda, T., Supasa,S., et al. (2010). Cross-reacting antibodies enhance dengue virusinfection in humans. Science 328, 745-748). K562 cells werepre-incubated with serial dilutions of prM cross-reactive antibody DV62(Beltramello, M., Williams, K. L., Simmons, C. P., Macagno, A.,Simonelli, L., Quyen, N. T. H., Sukupolvi-Petty, S., Navarro-Sanchez,E., Young, P. R., de Silva, A. M., et al. (2010). The human immuneresponse to Dengue virus is dominated by highly cross-reactiveantibodies endowed with neutralizing and enhancing activity. Cell HostMicrobe 8, 271-283) derived from a DENV immune donor. Results are shownin FIG. 16D. DV62 cross-reacted with ZIKV prM protein and caused ADE ata broad range of concentrations (FIG. 16D). ZKA190-LALA can fully blockanti-prM DV62 mAb-induced ADE of immature or partially immature ZIKVparticles (FIG. 16D).

Finally, the ability of different concentrations of ZKA190, ZKA190-LALAand ZKA190 Fab to cause or block ADE of ZIKV in the presence ofenhancing concentrations of human anti-DENV2 plasma or DV62 was tested.Results are shown in FIG. 16E. ZKA190 at low concentrations increasedthe prM DV62-mediated ADE of ZIKV infection, consistent with its abilityto promote the entry of both immature and mature virions, while atconcentrations above 1.3 nM (i.e., 200 ng/ml) ZKA190 blocked ADE inducedby both DENV plasma and mAb DV62. ZKA190-LALA, as well as its Fabfragment, reduced ADE at concentrations above 0.06 nM, indicating thatboth inhibited virus infection at a post-attachment step, such asfusion.

Example 10: ZKA190 Binds to a Conserved and Highly Accessible Region ofEDIII

To determine the ZKA190 epitope at the residue level, solution NMRspectroscopy was used as described in Bardelli, M., Livoti, E.,Simonelli, L., Pedotti, M., Moraes, A., Valente, A. P., and Varani, L.(2015). Epitope mapping by solution NMR spectroscopy. J. Mol. Recognit.28, 393-400; Simonelli, L., Beltramello, M., Yudina, Z., Macagno, A.,Calzolai, L., and Varani, L. (2010). Rapid structural characterizationof human antibody-antigen complexes through experimentally validatedcomputational docking. J Mol Biol 396, 1491-1507; and Simonelli, L.,Pedotti, M., Beltramello, M., Livoti, E., Calzolai, L., Sallusto, F.,Lanzavecchia, A., and Varani, L. (2013). Rational Engineering of a HumanAnti-Dengue Antibody through Experimentally Validated ComputationalDocking. PLoS ONE 8, e55561.

Briefly, spectra were recorded on a Bruker Avance 700 MHz NMRspectrometer at 300 K. For assignments of backbone resonances standardtriple resonance experiments (HNCO, HN(CA)CO, HN(CO)CACB, HNCACB wereused, while sidechains were annotated using HCCH-TOCSY and HBHA(CO)NHexperiments. All NMR experiments were processed using Topspin 2.1(Bruker Biospin) and analysed with CARA. NOESY cross peaks wereautomatically assigned using the CYANA “noeassign” macro based on themanually assigned chemical shifts. Upper-distance restraints used forthe structure calculations in CYANA using the standard simulatedannealing protocol were derived from 70 ms ¹⁵N- and ¹³C-resolved NOESYspectra. Backbone dynamics of ZIKV EDIII were derived from ¹⁵Nrelaxation measurements recorded on 600 and 700 MHz spectrometers.Proton-detected versions of the CPMG (R2), inversion-recovery (R1) and¹⁵N {¹H}-steady-state NOE were utilized. Delay settings for the T2series were in the range of 0 to 0.25 sec and for the T1 series between0.02 to 2 sec. The ¹⁵N {¹H}-NOE experiment used a relaxation delay of 5s. The R1 and R2 relaxation rates were derived from least-squares fitsof corresponding exponential functions to the measured data usinghome-written scripts. The relaxation data were analyzed in a model-freeapproach using the software package DYNAMICS. The program ROTDIF wasused to calculate the overall correlation time from the relaxation data(8.5 ns). NMR epitope mapping was performed as previously described(Bardelli et al., 2015; Simonelli et al., 2010; 2013). Briefly, overlayof ¹⁵NHSQC spectra of labelled EDIII free or bound to ZKA190 Fab allowedidentification of EDIII residues whose NMR signal changed upon complexformation, indicating that they were affected by ZKA190 binding. Changeswere identified by manual inspection and by the Chemical ShiftPerturbation (CSP), CSP=((Δδ_(H))²±(Δδ_(N)/10)²)^(1/2). NMR samples weretypically 800 μM of

¹⁵N, ¹³C

-labeled EDIII in 20 mM sodium phosphate, 50 mM NaCl, pH 6.0.Perdeuterated (nominally 70%) ²H,¹⁵N EDIII samples were used for NMRepitope mapping with a EDIII:ZKA190 Fab ratio of 1:1.1; EDIIIconcentration was typically 0.4 mM.

Since the NMR signal is strongly dependent on the local chemicalenvironment, changes upon complex formation identify antigen residuesthat are affected by antibody binding, either directly or throughallosteric effects. By comparing the NMR spectra of free and bound EDIII(FIG. 17A), residues affected by ZKA190 were mapped to the LR of EDIII,in particular to the BC, DE and FG loops, as well as to part of theEDI-EDIII hinge (FIG. 18A). These residues are nearly identical among217 known ZIKV strains, with the exception of substitutions at V341I andE393D in the Uganda 1947 isolate (FIG. 17D). These mutations are alsopresent in the MR766 strain that was efficiently neutralized by ZKA190(FIG. 15A). Analysis of the ZKA190 epitope on the uncomplexed ZIKVstructure showed that the epitope is highly accessible, except for theFG loop in the 5-fold vertex (FIGS. 18B and 17C, molecule A).

Computational docking followed by molecular dynamics simulation, guidedand validated by NMR-derived epitope information as well as EDIIImutagenesis, showed that ZKA190 binds through an interface characterizedby shape and charge complementarity (FIGS. 18B and 17E). Dockingindicates that there are no direct contacts between ZKA190 and the FGloop on EDIII, suggesting that changes in its NMR signals upon antibodybinding derive from allosteric effects. This notion is supported by thefact that mutations of FG loop residues in recombinant EDIII, but not inother epitope regions, did not affect the binding affinity of ZKA190 forEDIII (FIGS. 18B and 19).

Example 11: Mechanisms of ZKA190 Neutralization

The ability of ZKA190 to efficiently neutralize the virus may involveinhibition of either cell attachment or membrane fusion. A furthermechanism might involve virus inactivation through cross-linking ofviral particles.

ZKA190 Fab can neutralize ZIKV, albeit less efficiently than thecorresponding IgG. By binding to the EDI-EDIII linker, ZKA190 (both Faband IgG) might inhibit the ˜70 degree rotation of DIII required forviral fusion to the host cell membrane (Bressanelli, S., Stiasny, K.,Allison, S. L., Stura, E. A., Duquerroy, S., Lescar, J., Heinz, F. X.,and Rey, F. A. (2004). Structure of a flavivirus envelope glycoproteinin its low-pH-induced membrane fusion conformation. Embo J 23, 728-738;Modis, Y., Ogata, S., Clements, D., and Harrison, S. C. (2004).Structure of the dengue virus envelope protein after membrane fusion.Nature 427, 313-319). Alternatively, ZKA190 might prevent the attachmentof ZIKV to target cells.

The ability of ZKA190 to inhibit membrane fusion is supported byconfocal microscopy analysis. To this end, Vero cells were plated at7,500 cells/well on 12 mm-diameter coverslips in 24-well plates andincubated overnight. Cells were infected with ZIKV H/PF/2013 (MOI of100) in the presence or absence of neutralizing concentrations ofAlexa-488 conjugated mAbs (0.7 μM) at 37° C. for 3 h, washed with PBS,and fixed with 2% paraformaldehyde in PBS for 30 min at roomtemperature. Acidified endosome were identified with Lysotracker red(Invitrogen) by adding the dye (50 nM) to the cells for the last 30 minof the incubation prior to fixation. Fixation was followed by extensivewashes in PBS and 50 mM glycine and finally the coverslips were preparedfor microscopy analysis using Vectashield mounting medium forfluorescence with DAPI (Vector Laboratories). Samples were analyzed byconfocal microscopy using a Leica TCS SP5 microscope with a 63×/1.4 N.A.objective. Image analysis and processing was performed with FIJIsoftware.

Results are shown in FIG. 20. Confocal microscopy analysis shows thatZKA190 (Fab or IgG) can enter Vero cells only when complexed with ZIKV,at neutralizing concentrations exceeding the IC50 by 10,000-fold (FIG.20).

Example 12: In Vivo Characterization of the EDIII-Specific mAb ZKA190

To evaluate their prophylactic and therapeutic properties, ZKA190 andZKA190-LALA were tested in A129 mice challenged with a lethal dose ofZIKV strain MP1751 (African lineage). To test their prophylacticpotencies, ZKA190 and ZKA190-LALA were administered one day before viruschallenge.

Female A129 mice (IFN-alpha/beta receptor −/−) and wild-type 129Sv/Evmice aged 5-8 weeks were administered mAbs (ZKA190, ZKA190-LALA andcontrol antibody MPE8 (Corti, D., et al. Cross-neutralization of fourparamyxoviruses by a human monoclonal antibody. Nature 501, 439-443(2013)) diluted in PBS at different doses via the intraperitoneal (i.p.)route in a volume of 500 μl. MAbs were administered either 1 day beforeor 1, 2, 3 or 4 days after virus challenge. Animals were challengedsubcutaneously with 102 pfu ZIKV (strain MP1751) and followed for 14days. Weights and temperatures were monitored daily and clinicalobservations were recorded at least twice per day. On day 5post-challenge, 50 μl of blood was collected from each animal into aRNAprotect tube (Qiagen, UK) and frozen at −80° C. At the end of thestudy (14 days post-challenge) or when animals met human endpoints,necropsies were undertaken, and blood and sections of brain, spleen,liver, kidney and ovary were collected for virological analysis.

Tissue samples from A129 mice were weighed and homogenized into PBSusing ceramic beads and an automated homogenizer (Precellys, UK) usingsix 5 second cycles of 6500 rpm with a 30 second gap. Two hundred μl oftissue homogenate or blood solution was transferred into 600 μL RLTbuffer (Qiagen, UK) for RNA extraction using the RNeasy Mini extractionkit (Qiagen, UK); samples were passed through a QIAshredder (Qiagen, UK)as an initial step. A ZIKV specific realtime RT-PCR assay was utilizedfor the detection of viral RNA from subject animals. The primer andprobe sequences were adopted from Quick et al., 2017 (Quick, J, GrubaughN D, Pullan S T, Claro I M, Smith A D, Gangavarapu K, Oliveira G,Robles-Sikisaka R, Rogers T F, Beutler N A, et al.: Multiplex PCR methodfor MinION and Illumina sequencing of Zika and other virus genomesdirectly from clinical samples. Nat Protoc 2017, 12:1261-1276) within-house optimization and validation performed to provide optimalmastermix and cycling conditions. Real-time RT-PCR was performed usingthe SuperScript III Platinum One-step qRT-PCR kit (Life Technologies,UK). The fmal mastermix (15 μl) was comprised of 10 μl of 2× ReactionMix, 1.2 μl of PCR-grade water, 0.2 μl of 50 mM MgSO4, 1 μl of eachprimer (ZIKV 1086 and ZIKV 1162c both at 18 μM working concentration),0.8 μl of probe (ZIKV 1107-FAM at 25 μM working concentration) and 0.8μl of SSIII enzyme mix. Five μl of template RNA was added to themastermix, yielding a final reaction volume of 20 μl. The cyclingconditions used were 50° C. for 10 minutes, 95° C. for 2 minutes,followed by 45 cycles of 95° C. for 10 seconds and 60° C. for 40seconds, plus a final cooling step of 40° C. for 30 seconds.Quantification analysis using fluorescence was performed at the end ofeach 60° C. step. Reactions were run and analyzed on the 7500 Fastplatform (Life Technologies, UK) using 7500 software version 2.0.6.Quantification of viral load in samples was performed using a dilutionseries of quantified RNA oligonucleotide (Integrated DNA Technologies).The oligonucleotide comprised the 77 bases of ZIKV RNA targeted by theassay, based on GenBank accession AY632535.2 and was synthesized to ascale of 250 nmole with HPLC purification.

Results are shown in FIGS. 21, 22 and 23. ZKA190 and ZKA190-LALA wereshown to protect mice from mortality and morbidity at concentrations of5, 1 or 0.2 mg/kg (FIG. 21A-B). ZKA190-LALA, and to a lesser extentZKA190, delayed morbidity and mortality as compared to the control groupat 0.04 mg/kg. Viral titers in blood and organs were reducedsignificantly compared to control antibody-treated animals, even in thepresence of serum antibody levels below 1 μg/ml (FIG. 22A-D).

To evaluate the therapeutic potential of ZKA190, we administered ZKA190and ZKA190-LALA at different time-points following ZIKV infection. At adose of 15 mg/kg, survival rates of 80%-100% were achieved, and themorbidity was greatly reduced even when treatment was given four dayspost-infection (FIG. 21E-G). ZKA190 and ZKA190-LALA treatment at allpost-infection time-points resulted in significantly reduced viraltiters, compared to animals treated with control antibody, with a cleartrend for greater reduction with earlier treatment (FIG. 23A-21C). Ofnote, ZKA190-LALA showed a significantly reduced antiviral activity inthe blood day 5 sample as compared to ZKA190 when mAbs were given fourdays post-infection, a result that might be related to the impairedability of the LALA variant to facilitate rapid clearance of coatedvirions.

Tables of Sequences and SEQ ID Numbers

ZKA190 SEQ ID NO. Amino acid sequence CDRH₁ 1 GFTFSKYG CDRH₂ 2 ISYEGSNKCDRH₃ 3 AKSGTQYYDTTGYEYRGLEYFGY CDRL₁ 4 QSVSSSY CDRL₂ 5 DAS CDRL₂ 6LIYDASSRA long CDRL₃ 7 QQYGRSRWT VH 8QVQLVESGGGVVQPGRSLRLSCAASGFTFSKYGMHWVRQAPGKGLEWVAVISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTQYYDTTGYEYRGLEYFGYW GQGTLVTVSS VL 9EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKRGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGRSRWTFGQGTKVE1KZKA190 SEQ ID NO. Nucleic acid sequence CDRH₁ 10ggattcaccttcagtaaatatggc CDRH₂ 11 atatcatatgagggaagtaataaa CDRH₃ 12gcgaaatcggggacccaatactatgatactactggttatg agtataggggtttggaatactttggctacCDRL₁ 13 cagagtgttagtagcagttac CDRL₂ 14 gatgcatcc CDRL₂ 15ctcatctatgatgcatccagcagggcc long CDRL₃ 16 cagcagtatggtaggtcaaggtggaca VH17 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtaaatatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatcatatgagggaagtaataaatattatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggcagtgtattactgtgcgaaatcggggacccaatactatgatactactggttatgagtataggggtttggaatactttggctactgg ggccagggaaccctggtcaccgtctcctcagVL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagtagcagttacttagcctggtaccagcagaaacgtggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtaggtcaaggtggacattcggccaagggaccaaggtggaaat caaac ZKA185 SEQ ID NO.Amino acid sequence CDRH₁ 19 GYSFTSYW CDRH₂ 20 FDPSDSQT CDRH₃ 21ARRYCSSSSCYVDN CDRL₁ 22 ALPNKF CDRL₂ 23 EDN CDRL₂ 24 VIYEDNKRP longCDRL₃ 25 YSTDSSSNPLGV VH 26 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWITWVRQMPGKGLEWMAKFDPSDSQTNYSPSFQGHVTISVDKSISTAYLQWSSLKASDTAMYYCARRYCSSSSCYVDNWGQGTLVTIF S VL 27SYELTQPPSVSVSPGQTARITCSGDALPNKFAYWYRQKSGQAPVLVIYEDNKRPSGIPERFSGSSSGTMATLTISGAQVE DEADYHCYSTDSSSNPLGVFGGGTKLTVLZKA185 SEQ ID NO. Nucleic acid sequence CDRH₁ 28ggatatagttttaccagttactgg CDRH₂ 29 tttgatcctagtgactctcaaacc CDRH₃ 30gcgagaagatattgtagtagtagtagttgttatgtggaca at CDRL₁ 31 gcattgccaaataaatttCDRL₂ 32 gaggacaac CDRL₂ 33 gtcatctatgaggacaacaaacgaccc long CDRL₃ 34tactcaacagacagcagttctaatcccctgggagta VH 35gaagtgcagctggtgcagtccggagcagaggtgaaaaagcccggggagtctctgaggatctcctgtaagggttctggatatagttttaccagttactggatcacctgggtgcgccagatgcccgggaaaggcctggagtggatggcgaagtttgatcctagtgactctcaaaccaactacagcccgtccttccaaggccacgtcaccatctcagttgacaagtccatcagcactgcctacttgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagaagatattgtagtagtagtagttgttatgtggacaattggggccagggaaccctggtcaccatcttc tcag VL 36tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacaaacggccaggatcacctgctctggagatgcattgccaaataaatttgcttattggtaccggcagaagtcaggccaggcccctgttctggtcatctatgaggacaacaaacgaccctccgggatccctgagagattctctggctccagctcagggacaatggccaccttgactatcagtggggcccaggtggaggatgaagctgactaccactgttactcaacagacagcagttctaatcccctgggagtattcggcggagggaccaagctgac cgtcctag ZKA230 SEQ ID NO.Amino acid sequence CDRH₁ 37 GGSISSDY CDRH₂ 38 IYYSGST CDRH₃ 39ARRRKYDSLWGSFAFDI CDRL₁ 40 SSNIGGNY CDRL₂ 41 IND CDRL₂ 42 LICINDHRP longCDRL₃ 43 ATWDDSLGGLV VH 44 QVQLQESGPGLVKPSETLSLTCAVSGGSISSDYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNHFSLKLNSVTAADTAVYYCARRRKYDSLWGSFAFDIWGQGTMVT VSS VL 45QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNYVYWYQQLPGTAPKLLICINDHRPSGVPDRFSGSKSGTSASLAISGLQ SEDEADYYCATWDDSLGGLVFGGGTKLTVLZKA230 SEQ ID NO. Nucleic acid sequence CDRH₁ 46ggtggctccatcagtagtgactac CDRH₂ 47 atctattacagtgggagcacc CDRH₃ 48gcgaggaggaggaagtatgattccctttgggggagttttg cttttgatatc CDRLi 49agctccaacatcggaggtaattat CDRL₂ 50 attaatgat CDRL₂ 51ctcatctgtattaatgatcaccggccc long CDRL₃ 52gcaacatgggatgacagcctgggtggccttgta VH 53caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcggagaccctgtccctcacctgcgcagtctctggtggctccatcagtagtgactactggagctggatccggcagcccccagggaagggactggagtggattgggtatatctattacagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccacttctccctgaagctgaactctgtgaccgctgcggacacggccgtgtattactgtgcgaggaggaggaagtatgattccctttgggggagttttgcttttgatatctggggccaagggacaatggtcacc gtctcttcag VL 54cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaggtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcctcatctgtattaatgatcaccggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtccgaggatgaggctgattattactgtgcaacatgggatgacagcctgggtggccttgtattcggcggagggaccaagct gaccgtcctag ZKA78 SEQ ID NO.Amino acid sequence CDRH₁ 55 GFTFSNYA CDRH₂ 56 IGRNGDSI CDRH₃ 57VKDLAIPESYRIEADY CDRL₁ 58 QSVLYRSNNKNY CDRL₂ 59 WAS CDRL₂ 60 LIYWASTRElong CDRL₃ 61 QQYYSSPRT VH 62 EVQLAESGGGLVQPGGSLTLSCSGSGFTFSNYAMVWARQAPGKGLEYVSGIGRNGDSIYYTDSVKGRFTISRDNSKSMVYLQMSSLRTEDTAVYYCVKDLAIPESYRIEADYWGQGTLVI VSA VL 63DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLSWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISPLQAEDVAVYYCQQYYSSPRTFGQGTKVE1K ZKA78 SEQ ID NO. Nucleic acid sequenceCDRH₁ 64 ggcttcacttttagtaactatgca CDRH₂ 65 atcgggcgcaacggggactctatcCDRH₃ 66 gtgaaagatctggccatccccgagtcctacagaattgaag ctgattat CDRL₁ 67cagtccgtgctgtaccgctctaacaacaagaattac CDRL₂ 68 tgggcttca CDRL₂ 69ctgatctattgggcttcaacccgggaa long CDRL₃ 70 cagcagtactattctagtcctcgaact VH71 gaggtgcagctggcagaatcaggcgggggactggtccagcctggcggcagcctgacactgtcttgcagtggatcaggcttcacttttagtaactatgcaatggtgtgggcaaggcaggctcctgggaagggactggagtatgtctctggcatcgggcgcaacggggactctatctactatactgatagtgtgaagggccggttcaccatcagcagagacaatagcaaatccatggtgtacctgcagatgagctccctgcgaaccgaagacacagcagtgtactattgcgtgaaagatctggccatccccgagtcctacagaattgaagctgattattggggacagggcaccctggtcatc gtgagcgccg VL 72gacatcgtgatgacacagtctccagatagtctggcagtcagtctgggggagagggccactattaactgcaagagctcccagtccgtgctgtaccgctctaacaacaagaattacctgtcttggtatcagcagaagcccggacagccccctaaactgctgatctattgggcttcaacccgggaaagcggcgtcccagacagattctcaggcagcgggtccggaacagacttcaccctgacaattagccccctgcaggcagaggacgtggctgtctactattgtcagcagtactattctagtcctcgaactttcggccaggg gaccaaggtggaaatcaaac ZKA64SEQ ID NO. Amino acid sequence CDRH₁ 73 GYTFTGYH CDRH₂ 74 INPNSGGT CDRH₃75 ARMSSSIWGFDH CDRL₁ 76 QSVLIN CDRL₂ 77 GAS CDRL₂ 78 LIYGASSRA longCDRL₃ 79 QQYNDWPPIT VH 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHIDWVRQARGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMQLSRLRSDDSAVYYCARMSSSIWGFDHWGQGTLVTVSS VL 81EIVMTQSPATLSVSPGERATLSCRASQSVLINLAWYQQKPGQAPRLLIYGASSRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNDWPPITFGQGTRLE1KZKA64 SEQ ID NO. Nucleic acid sequence CDRH₁ 82 ggctacaccttcacagggtatcacCDRH₂ 83 attaaccctaattctggcgggacc CDRH₃ 84gctcggatgagctcctctatttggggcttcgatcat CDRL₁ 85 cagtctgtgctgattaac CDRL₂86 ggagcatcc CDRL₂ 87 ctgatctatggagcatcctccagggct long CDRL₃ 88cagcagtacaatgattggccccctatcaca VH 89caggtgcagctggtccagagcggagcagaggtgaagaaacccggcgcctcagtgaaggtcagctgcaaagcttccggctacaccttcacagggtatcacatcgactgggtgaggcaggcaagaggacagggactggaatggatgggacggattaaccctaattctggcgggaccaactacgcccagaagtttcagggccgagtgactatgaccagagacaccagcatctccacagcttatatgcagctgtcccggctgagatctgacgatagtgccgtctactattgtgctcggatgagctcctctatttggggcttcgatcattgggggcagggaacactggtgactgtcagttcag VL 90gagatcgtgatgactcagtctccagccaccctgtcagtcagcccaggagaacgggcaaccctgtcttgcagagcctcccagtctgtgctgattaacctggcttggtaccagcagaagccaggccaggcaccccgactgctgatctatggagcatcctccagggctaccggcattcctgcacgcttcagtggatcaggaagcggaacagagtttaccctgacaatctctagtctgcagtccgaagacttcgctgtctactattgtcagcagtacaatgattggccccctatcacatttggccaggggactagactggagat caagc ZKA15 SEQ ID NO.Amino acid sequence CDRH₁ 91 GG F I N SYY CDRH₂ 92 IY K SGST CDRH₃ 93ARDPYGDYVKAFDI CDRL₁ 94 QSLLHSNGYNY CDRL₂ 95 LGS CDRL₂ 96 LIYLGSNRA longCDRL₃ 97 MQALQTVT VH 98 QVQLQESGPGLVKPSETLSLTCTVSGG F I N SYYWSWIRQPAGKGLEWIGRIY K SGSTNYNPSLKSRVTMSLDTSKYQFSLKLRSVTAADTAVYYCARDPYGDYVKAFDIWGQGTMVTVSS VL 99DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLNWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCMQALQTVTFGPGTKVD1KZKA15 SEQ ID NO. Nucleic acid sequence CDRH₁ 100ggtggcttcatcaatagttactac CDRH₂ 101 atctataaaagtgggagcacc CDRH₃ 102gcgagagatccctacggtgactacgttaaggcttttgata tt CDRL₁ 103cagagcctcctgcatagtaatggatacaactat CDRL₂ 104 ttgggttct CDRL₂ 105ctgatctatttgggttctaatcgggcc long CDRL₃ 106 atgcaagctctacaaactgtcact VH107 caggtgcagctgcaggagtcggggccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctccggtggcttcatcaatagttactactggagctggatccggcagcccgccgggaagggactggagtggattgggcgtatctataaaagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatgtcactagacacgtccaagtaccagttctccctgaagctgaggtctgtgaccgccgctgacacggccgtgtattactgtgcgagagatccctacggtgactacgttaaggcttttgatatttggggccaagggacaatggtcaccgtctcttca g VL 108gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtaatggatacaactatttgaattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactgtcactttcggccctgggaccaa agtggatatcaaac ZKA25 SEQ ID NO.Amino acid sequence CDRH₁ 109 GFTF R S H W CDRH₂ 110 IK E DG Y EK CDRH₃111 ARDLRVYSGRGFDP CDRL₁ 112 KLGDKY CDRL₂ 113 QDS CDRL₂ 114 VIYQDSKRPlong CDRL₃ 115 QAWDSSTVV VH 116 EVQLVESGGGLVRPGGSLRLSCAASGFTF R S HWMSWVRQA PGKGLEWVANIK E DG Y EKYYVDSVKGRFTISRDNAKNSLYLQMKSLRAEDTAVYYCARDLRVYSGRGFDPWGQGTLVTVS S VL 117SYELTQPPSLSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPARFSGSNSGNTATLTISGTQAM DEADYYCQAWDSSTVVFGGGTKLTVLZKA25 SEQ ID NO. Nucleic acid sequence CDRH₁ 118ggattcacctttagaagtcattgg CDRH₂ 119 ataaaggaagatggatatgagaaa CDRH₃ 120gcgagagatttgagggtatatagtgggagaggtttcgacc cc CDRL₁ 121 aaattgggggataaatatCDRL₂ 122 caagatagc CDRL₂ 123 gtcatctatcaagatagcaagcggccc long CDRL₃ 124caggcgtgggacagcagcactgtggta VH 125gaggtgcagttggtggagtctgggggaggcttggtccggcctggggggtccctgagactctcctgtgcagcctctggattcacctttagaagtcattggatgagttgggtccgccaggctccagggaaggggctggagtgggtggccaacataaaggaagatggatatgagaaatactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaagagcctgagagccgaggacacggccgtgtattactgtgcgagagatttgagggtatatagtgggagaggtttcgacccctggggccagggaaccctggtcaccgtctcc tcag VL 126tcctatgagctgactcagccaccctcactgtccgtgtccccaggacagacagccagcatcacctgctctggagataaattgggggataaatatgcttgctggtatcagcagaagccaggccagtcccctgtgttggtcatctatcaagatagcaagcggccctcagggatccctgcgcgattctctggctccaactctgggaacacagccactctgaccatcagcgggacccaggctatggatgaggctgactattactgtcaggcgtgggacagcagcactgtggtattcggtggagggaccaagctgaccgtcctag ZKA35 SEQ ID NO.Amino acid sequence CDRH₁ 127 GGSIS T GGYY CDRH₂ 128 IYYSG N T CDRH₃ 129A K GGG R ERPFDY CDRL₁ 130 SSNIG R NY CDRL₂ 131 RNN CDRL₂ 132 LIYRNNQRPlong CDRL₃ 133 V AWDDS R SGFVV VH 134 QVQLQESGPGLVKPSQTLSLTCTVSGGSIS TGGYYWSWIR QHPGKGLEWIGYIYYSG N TYYNPSLKSRVTISVDTSKKQF SLKLSSVTAADTAVYYCAK GGG R ERPFDYWGQGTLVTVSS VL 135 QSVLTQPPSASGTPGQRVTISCSGSSSNIG RNYVDWYQQL PGTAPKLLIYRNNQRPSGVPERFSGSKSGTSASLAISGLR SEDEADYYC V AWDDS RSGFVVFGGGTKVTVL ZKA35 SEQ ID NO. Nucleic acid sequence CDRH₁ 136ggtggctccatcagcactggtggttactac CDRH₂ 137 atctattacagtgggaacacc CDRH₃ 138gcgaaaggaggagggagggagcgaccctttgactac CDRL₁ 139 agctccaacatcggaagaaattatCDRL₂ 140 aggaataat CDRL₂ 141 ctcatctataggaataatcagcggccc long CDRL₃ 142gtagcatgggatgacagccggagtggttttgtggta VH 143caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcactggtggttactactggagctggatccgccagcacccagggaagggcctggagtggattggttacatctattacagtgggaacacctactacaacccgtccctcaagagtcgagttaccatatcagttgacacctctaagaagcagttctccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgaaaggaggagggagggagcgaccctttgactactggggccagggaaccctggtcaccgtctcctca g VL 144cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagaaattatgtagactggtaccagcaactcccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgagcgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgtagcatgggatgacagccggagtggttttgtggtattcggcggagggaccaa ggtgaccgtcctag Constant regionsSEQ ID NO. Sequence IgG₁ 145 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSCH₁-CH₂- WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT CH₃ aaYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK IgG₁ 146ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS CH₁-CH₂-WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT CH₃ LALAYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GG aaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK IgG 147RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ CK aaWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC IgG148 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV CL aaTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECSIgG₁ 149 gcgtcgaccaagggcccatcggtcttccccctggcaccct CH₁-CH₂-cctccaagagcacctctgggggcacagcggccctgggctg CH₃ nuclcctggtcaaggactacttccccgaacctgtgacggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaAgaCcctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtccccgggtaaaIgG₁ 150 gcgtcgaccaagggcccatcggtcttccccctggcaccct CH₁-CH₂-cctccaagagcacctctgggggcacagcggccctgggctg CH₃ LALAcctggtcaaggactacttccccgaacctgtgacggtctcg nucltggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaaGCCGCGgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtccccgggtaaaIgG CK 151 cgTacGgtggctgcaccatctgtcttcatcttcccgccat nuclctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtg t IgG CL 152ggtcagcccaaggctgccccctcggtcactctgttcccgc nuclcctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcttggaaagcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca SEQ ID NO. Amino acid sequenceZKA10 CDRH₁ 153 GFTFSDSY CDRH₂ 154 ISSSSPFT CDRH₃ 155 ARGLVRDGYKWLYFFDYVH 156 QVQLVESGGGLVEPRGSLRLSCAASGFTFSDSYMSWIRQAPGKGLEWISYISSSSPFTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLVRDGYKWLYFFDYWGQGTLV TVSS ZKA18 CDRH₁ 157 GFTFSSYGCDRH₂ 158 IWYDGSNK CDRH₃ 159 ARDDSGYSEPFDY VH 160QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTITRDNSKNTLYLQMNSLRPEDTAVYYCARDDSGYSEPFDYWGQGTLVTVSS ZKA28 CDRH₁ 161 GFTVSRNY CDRH₂162 IYSGGST CDRH₃ 163 ARWINDAFDI VH 164EVQLVESGGGLIQPGGSLRLSCAASGFTVSRNYMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWINDAFDIWGQGTMVTVSS ZKA29 CDRH₁ 165 GFTFSRYS CDRH₂ 166ISPRSTTI CDRH₃ 167 AREDCTNGVCYRVDY VH 168EVQLVESGGGLVQPGGSLRLSCVVSGFTFSRYSMNWVRQAPGKGLEWVSYISPRSTTIYYADSVEGRFTVSRDNAKNSLYLQLNSLRAEDTAVYYCAREDCTNGVCYRVDYWGQGTLVTV SS ZKA33 CDRH₁ 169 GFTFSRNWCDRH₂ 170 IKEDGNEK CDRH₃ 171 ARPFHQGGYAYGLAY VH 172EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNWMTWVRQAPGKGLEWVANIKEDGNEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPFHQGGYAYGLAYWGQGTLVTV SS ZKA39 CDRH₁ 173 GFTFSTYSCDRH₂ 174 ISPSSSTI CDRH₃ 175 AREYCSGGSCYLLDY VH 176EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSYISPSSSTIYYPDSLKGRFTISRDNAKNSLYLQMDSLRAEDTAQYYCAREYCSGGSCYLLDYWGQGTLVTV SS ZKA43 CDRH₁ 177 GGSITSYYCDRH₂ 178 SHYSGST CDRH₃ 179 ARGIYSGKNWFDP VH 180QVQLQESGPGLVKPSETLSLTCTVYGGSITSYYWTWIRQPPGKGLEWIGYSHYSGSTNYNPSLKSRVTISIDTSKSQFSLNLNSVTAADTAVYYCARGIYSGKNWFDPWGQGTLVTVSS ZKA44 CDRH₁ 181 GFTVSTSY CDRH₂182 IYSSGST CDRH₃ 183 ARVSLGGLDP VH 184EVQLVESGGGLIQPGGSLRLSCVASGFTVSTSYMNWVRQAPGKGLEWVSVIYSSGSTYYADSVKGRFTISRNTSKNTLYLQMNSLRAEDTAVYYCARVSLGGLDPWGQGTPVTVSS ZKA46 CDRH₁ 185 GFSLSNGRMG CDRH₂186 IFSNDEK CDRH₃ 187 ARVEFRAGNYLDS VH 188QVTLKESGPVLVKPTETLTLTCTVSGFSLSNGRMGVSWIRQPPGKALEWLAHIFSNDEKYYSTSLKNRLTISKDTSKSQVVLTMTNMDPVDTATYYCARVEFRAGNYLDSWGQGTLVTVS S ZKA50 CDRH₁ 189 GYTFTNSWCDRH₂ 190 IYPGDSDT CDRH₃ 191 ARQPFFDY VH 192EVQLVQSGAQVKKPGESLKISCKASGYTFTNSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQPFFDYWGQGTLVTVSS ZKA54 CDRH₁ 193 GYTFTGYY CDRH₂ 194INANSGGT CDRH₃ 195 AHSDIVVVPSDDYYALDV VH 196QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYYMHWVRQAPGQGLEWMGWINANSGGTNFAQRFQGRVTMTWDTSISTAYMELSRLRSDDTAVYYCAHSDIVVVPSDDYYALDVWGQGTT VTVSS ZKB18 CDRH₁ 197 GYSFTSYWCDRH₂ 198 IYPGDSDT CDRH₃ 199 ARQTPGDY VH 200EVQLVQSGAEVKKPGESLKISCKTFGYSFTSYWIGWVRQMPGKGLEWMGMIYPGDSDTRYSPSFQGQVTISADMSISTAYLQWSSLKASDTAMYYCARQTPGDYWGQGTLVTVSS ZKB20 CDRH₁ 201 GYFFTRYV CDRH₂ 202INTDNGST CDRH₃ 203 ARGTGRDGYNSFFAN VH 204QVQLVQSGAEVKKPGASVRVSCKASGYFFTRYVILWVRQAPGQRPEWMGWINTDNGSTRYSQKFQGRVTITKDTSATTAYMDLSSLKSDDTAVYYCARGTGRDGYNSFFANWGQGTLVTV SP ZKB21 CDRH₁ 205 GYTFTGYSCDRH₂ 206 IDTNSGDT CDRH₃ 207 ARDRERHPFSY VH 208QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYSIHWVRQAPGQGLAWMGRIDTNSGDTNYAERFQGRVTMTRDTSISTAYMEVRRLRSDDTAVYYCARDRERHPFSYWGQGTLVTVSS ZKB23 CDRH₁ 209 GGSISSGDYS CDRH₂210 ITHSGTT CDRH₃ 211 ARHFGWFDP VH 212QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGDYSWSWIRQPPGKGLEWIGYITHSGTTYFNPSLKSRVTISVDRSRNQFSLKVTSVTAADTAVYYCARHFGWFDPWGQGTLVTVSS ZKC29 CDRH₁ 213 GGSISSGEYF CDRH₂214 IHNRGNT CDRH₃ 215 ARGGGDLVVVPDSIWDYYGMDV VH 216QVQLQESGPGLVRPSQTLSLTCTVSGGSISSGEYFWTWIRQHPKKGLEWIGYIHNRGNTYYNPSLKSRLSISLDTSKNHLSLRLSSVTAADTAVYYCARGGGDINVVPDSIWDYYGMDVW GQGTTVTVSS ZKC31 CDRH₁ 217GGSISSGGYH CDRH₂ 218 IYYSGST CDRH₃ 219 ARDRSEPGEYHYYYYAMDV VH 220QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYHWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKRRVTISVDTSKNQFSLKLSSVSAADTAVYYCARDRSEPGEYHYYYYAMDVWGQG TTVTVSS ZKC32 CDRH₁ 221GFTVSSNY CDRH₂ 222 IYSSGST CDRH₃ 223 ARGKKGNAFDI VH 224EVQLVESGGDLIQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAGDTAVYYCARGKKGNAFDIWGQGTVVTVSS ZKC33 CDRH₁ 225 GDSISSRTFS CDRH₂226 IYYSGST CDRH₃ 227 ARRNAEFFSFWSYYGMDV VH 228QVQLQESGPGLVKPSQTLSLTCTVSGDSISSRTFSWSWIRQPPGKGLEWVGHIYYSGSTDYNPSLKSRISISIDTSKNQFSLKLSSVTAADTAVYYCARRNAEFFSFWSYYGMDVWGHGT AVIVSS ZKC34 CDRH₁ 229GGSINSGGYY CDRH₂ 230 ILHSGNT CDRH₃ 231 ARAGDYYSGYVPPEY VH 232QVQLQESGPGLVKPSQTLSLTCAVSGGSINSGGYYWSWVRQHPGKGLEWIGYILHSGNTNYNPSLKSRVNIFVDTSENQFSLKLRSVTAADTAIYFCARAGDYYSGYVPPEYWGPGTLVT VSS ZKD25 CDRH₁ 233 GFTVSSNYCDRH₂ 234 IYSGGST CDRH₃ 235 ARFGGNPSFDY VH 236EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYANSVKGRFTISRDKSKNTLYLQMNNLRAEDTAVYFCARFGGNPSFDYWGQGTLVTVSS ZKA3 CDRH₁ 237 GFIFSNYA CDRH₂ 238IGGKGDSI CDRH₃ 239 VKDLAVLESDRLEVDQ VH 240EVQLAESGGGLVQPGGSLRLSCSGSGFIFSNYAMVWARQAPGKGLEYVSGIGGKGDSIYHIDSVKGRFTISRDNSKRTVYLQMSRLRTEDTAVYYCVKDLAVLESDRLEVDQWGQGTLVI VSA ZKA4 CDRH₁ 241 GFTFSSYVCDRH₂ 242 TSYDGSNK CDRH₃ 243 ARGPVPYWSGESYSGAYFDF VH 244QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVTVTSYDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRGEDTAIYYCARGPVPYWSGESYSGAYFDFWGQG ILVTVSS ZKA5 CDRH₁ 245 GFTFSNYYCDRH₂ 246 MSSSETIK CDRH₃ 247 ARSGIETVAGSIDYYGMDV VH 248QVQLVESGGGLVKPGGSLRLSCAGSGFTFSNYYMTWIRQAPGKGLELVSYMSSSETIKYYADSVKGRFTISRDNAKNSLYLQMNSLRADDTARYYCARSGIETVAGSIDYYGMDVWGHGT PVTVSS ZKA6 CDRH₁ 249 DFTVSNYACDRH₂ 250 VSYDGSNK CDRH₃ 251 ATGVTMFQGAQTNAEYLHY VH 252QVHLVESGGGVVQPGRSLRLSCEASDFTVSNYAMHWVRQAPGKGLEWVAVVSYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCATGVTMFQGAQTNAEYLHYWGQGS LVTISS ZKA7 CDRH₁ 253 GFTFSRYGCDRH₂ 254 VSGDGSST CDRH₃ 255 VKDFWSGDQSLESDF VH 256EVQLVESGGGLVQPGGSLRLSCSASGFTFSRYGMVWARQAPGKGLEYLSGVSGDGSSTYYANSVKGRFTISRDNSKNTLYLHMSRLRDEDTAMYYCVKDFWSGDQSLESDFWGQGALVTV SS ZKA8 CDRH₁ 257 GFTFSAHACDRH₂ 258 ISRNEDYT CDRH₃ 259 VKDFGTSPQTDF VH 260DERLVESGGGLVQPGGSLRLVCSASGFTFSAHAMHWVRQPPGKGLEYVSTISRNEDYTYYADSVKGRFTISRDNSKNSLYLQMRRLRPEDTAIYYCVKDFGTSPQTDFWGQGTLVAVSS ZKA76 CDRH₁ 261 GFTFSTYF CDRH₂262 ISSTGSYK CDRH₃ 263 ARPFHSEYTYGLDAFDI VH 264EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYFMHWVRQAPGKGLEWVASISSTGSYKFYADSVKGRFTISRDNTKNSLFLQMNSLRAEDTAVFYCARPFHSEYTYGLDAFDIWGQGTML TVSS ZKA117 CDRH₁ 265GGSIRRTNSY CDRH₂ 266 ISYSGST CDRH₃ 267 ARLNDGSTVTTSSYFDY VH 268QLQLQESGPGLVKPSETLSLTCTVSGGSIRRTNSYWGWIRQTTGKGLQWIGSISYSGSTFYNPSLKSRVTISLDTSKDHFSLELSSVTAADTAIYYCARLNDGSTVTTSSYFDYWGQGTL VTVSS ZKB27 CDRH₁ 269 GYSFTSSWCDRH₂ 270 IDPSDSYT CDRH₃ 271 ARHDYSVSENGMDV VH 272EVQLVQSGAEVKKPGESLRISCKASGYSFTSSWINWVRQMPGKGLEWMGRIDPSDSYTTYNPSFQGHVTISVDKSIGTAYLQWNSLRASDTAMYYCARHDYSVSENGMDVWGQGTTVTVS S ZKB29 CDRH₁ 273 GFTFSSYTCDRH₂ 274 ISYDGSHK CDRH₃ 275 ARRSYSISCFDY VH 276QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVAVISYDGSHKFYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTALYYCARRSYSISCFDYWGQGTLVTISS ZKB34 CDRH₁ 277 GFTFSRSG CDRH₂278 VSYDGSNK CDRH₃ 279 AKDLTMVRGVHYYYYVMDV VH 280QVQLVESGGGVVQPGRSLRLSCAASGFTFSRSGMHWVRQAPGKGLEWVAVVSYDGSNKYYSDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAKDLTMVRGVHYYYYVMDVWGQGT TVTVSS ZKB39 CDRH₁ 281 GYTFDDYYCDRH₂ 282 INPHRGGT CDRH₃ 283 VRDQYCDGGNCYGIHQPHYGMDV VH 284QVQLVQSGAEVKKPGASLKVSCKASGYTFDDYYIHWVRQAPGQGLEWLGRINPHRGGTNYAQKFQGRVIMTLDMSISTTYMELRRITSDDAAVYYCVRDQYCDGGNCYGIHQPHYGMDVW GQGTTVTVSS ZKB46 CDRH₁ 285GYSFTSYW CDRH₂ 286 IDPSDSYT CDRH₃ 287 ARREYSSSSGQEDWFDP VH 288EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMPGKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARREYSSSSGQEDWFDPWGQGTLV TVSS ZKB53 CDRH₁ 289 GFTFSSYACDRH₂ 290 ISYDGSNR CDRH₃ 291 ARHVEQLPSSGYFQH VH 292QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQTPGKGLEWVTVISYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARHVEQLPSSGYFQHWGQGTLVTV SS ZKC26 CDRH₁ 293 GFIFSDFYCDRH₂ 294 IGHDGSYI CDRH₃ 295 ARAHGGFRH VH 296QVQVVESGGGLVKPGGSLRLSCAASGFIFSDFYMSWMRQAPGKGLEWVAYIGHDGSYILYADSVKGRFTISRDNAKNSLFLRMNSLRVEDTAVYYCARAHGGFRHWGQGTVVAVSP ZKD5 CDRH₁ 297 GFTFTSYG CDRH₂ 298ISYDGSNK CDRH₃ 299 ARDRDHYDLWNAYTFDY VH 300QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMHWVRQTPGKGLDWVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQMNSLRAADTALYYCARDRDHYDLWNAYTFDYWGQGTLV TVSS ZKDI CDRH₁ 301 GFTFSNYACDRH₂ 302 ISYDVSDK CDRH₃ 303 AGGPLGVVVIKPSNAEHFHH VH 304QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDVSDKYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAAYYCAGGPLGVVVIKPSNAEHFHHWGQG TLVTVSS ZKD8 CDRH₁ 305 GFTFINYACDRH₂ 306 ISYDGSNK CDRH₃ 307 ATDADAYGDSGANFHY VH 308QVQLVESGGGVVQPGKSLRLSCAASGFTFINYAIHWVRQAPGKGLEWVAVISYDGSNKFYTDSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCATDADAYGDSGANFHYWGQGTLVT VSS ZKD15 CDRH₁ 309 DASISSGGFSCDRH₂ 310 IYSSGDT CDRH₃ 311 ARAHTPTSKFYYYYAMDV VH 312QLQLQESGSGLVKPSQTLSLTCTVSDASISSGGFSWSWIRQPLGKGLEWLGYIYSSGDTFYNPSLQGRVTMSVDIFRSQFSLKLTSVTAADTAMYYCARAHTPTSKFYYYYAMDVWGQGT TVTVSS ZKD16 CDRH₁ 313 GFTFSDHFCDRH₂ 314 SRNKPNSYTT CDRH₃ 315 AKVGGCYGGDCHVENDY VH 316EVQLVESGGDLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLEWVGRSRNKPNSYTTEYAASVKGRFSISRDDSKKALYLQMNSLQTEDTAVYYCAKVGGCYGGDCHVENDYWGQGT LVTVSS ZKD17 CDRH₁ 317 GFIFSDYACDRH₂ 318 ISYDGSSR CDRH₃ 319 ARGYCSSGTCFSTNAEYFHP VH 320QVQMVESGGGVVQPGTSLRLSCATSGFIFSDYAMHWVRQAPGKGLEWVAVISYDGSSRLYADSVKGRFTVSRDNSKNTLYLQMHSLRAGDTAVYYCARGYCSSGTCFSTNAEYFHPWGQG TLATISS ZKD20 CDRH₁ 321GFTFSDHF CDRH₂ 322 SRNKPNSYTT CDRH₃ 323 ARVGGCNGGDCHVENDY VH 324EVQLVESGGGLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLEWVGRSRNKPNSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLQTEDTAVYYCARVGGCNGGDCHVENDYWGQGT LVTVSS ZKA134 CDRH₁ 325GGTFSAYA CDRH₂ 326 IIPFFGTA CDRH₃ 327 ARSDIVSTTRGYHHYGMDV VH 328QVHLVQSGAEVKKPGSSVNVSCKASGGTFSAYAISWVRQAPGQGLEWMGGIIPFFGTAYYAQKFKGRVTVTADKSTSTVYMEMTSLRSEDTAVYYCARSDIVSTTRGYHHYGMDVWGQGT TVTVSS ZKA246 CDRH₁ 329GYTFSDYY CDRH₂ 330 INPYSGGT CDRH₃ 331 ARGFTMISDREFDP VH 332QVQLVQSGAEVKRPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGRINPYSGGTNYAQKFHGRVTVTRDTSISTVYMELRGLRSDDTAVYYCARGFTMISDREFDPWGQGTLVTVS S ZKA256 CDRH₁ 333 GFTFSTYWCDRH₂ 334 IKQDGSEK CDRH₃ 335 ARDPGYDDFWSGSYSGSFDI VH 336EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNTKNSLYLQVNSLRAEDTAIYYCARDPGYDDFWSGSYSGSFDIWGQG TMVTVSS ZKB42 CDRH₁ 337GFTFNNYG CDRH₂ 338 ISYDGNKK CDRH₃ 339 VKYGERINGYSDPFDH VH 340QVQVVESGGGVVQPGRSLRLFCAASGFTFNNYGMHWVRQAPGKGLEWVALISYDGNKKYYADSVKGRFSISRDNSKNTLYLQMNRLRSGDTAVYHCVKYGERINGYSDPFDHWGQGTLVT VSS ZKB85 CDRH₁ 341 GYTFTTYACDRH₂ 342 INTNTGNP CDRH₃ 343 ARVIVPYAFDI VH 344QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAPGQGPEWVGWINTNTGNPTYAQGFTGRFVLSLDTSVSTAFLQISSLKAEDTAVYYCARVIVPYAFDIWGQGTMVTVSS ZKB47 CDRH₁ 345 GYTFTNYY CDRH₂346 INPSGGPT CDRH₃ 347 ARDQYGGYARYGMDV VH 348QVQLVQSGAEVKKPGASVKVSCQASGYTFTNYYMHWVRQAPGQGLEWMGIINPSGGPTSYAQKFQGRVTMTTDTSTSTVYMELSSLRSEDTAVYYCARDQYGGYARYGMDVWGQGTTVTV SS ZKC6 CDRH₁ 349 GYTFTGYYCDRH₂ 350 INPNSGGT CDRH₃ 351 ARVSDWGFAFDI VH 352QVQLVQSGTEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCARVSDWGFAFDIWGQGTMVTVSQ ZKA160 CDRH₁ 353 GGSITSYS CDRH₂354 IFYSGST CDRH₃ 355 ARDQTMPVWVGGMDV VH 356QVQLQESGPGLVKPSETLSLTCTVSGGSITSYSWSWIRQPPGKGLEWIGYIFYSGSTDYNPSLKSRVTISVDTSKDQFSLRLRSVTAADTAVYYCARDQTMPVWVGGMDVWGQGTTVTVS S ZKA172 CDRH₁ 357 GYIFTRYWCDRH₂ 358 IDPSDSYT CDRH₃ 359 ARQETAREDGMAV VH 360EVQLVQSGAEVKKPGKSLRISCKGSGYIFTRYWISWVRQMPGKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARQETAREDGMAVWGQGTTVIVSS ZKA174 CDRH₁ 361 GGSMSNSYYHCDRH₂ 362 IYYSGST CDRH₃ 363 ARNPVFNPLTLTHDAFDI VH 364QLQLQESGPGLVKPSETLSLTCTVSGGSMSNSYYHWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARNPVFNPLTLTHDAFDIWGQGT MVTVSS ZKA189 CDRH₁ 365GFTFSSYA CDRH₂ 366 ISGSGDNT CDRH₃ 367 AKWPYYDFWSGSESYFDP VH 368GVQLLESGGALVQPGKSLRLSCAASGFTFSSYALTWVRQAPGKGLQWVSAISGSGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWPYYDFWSGSESYFDPWGQGTL VTVSS ZKA195 CDRH₁ 369 GYNFPSYWCDRH₂ 370 IDPSDSYT CDRH₃ 371 ARADCRSTSCYLVFE VH 372EVQLVQSGAEVKKPGESLRISCKDSGYNFPSYWIHWVRQMPGKGLEWMGTIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARADCRSTSCYLVFEGQGTLVTVS S ZKA215 CDRH₁ 373 GYTFTSYWCDRH₂ 374 IDPSDSHT CDRH₃ 375 ARHALPNYFDS VH 376EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWISWVRQMPGKGLEWMGRIDPSDSHTDYSPSFQGHVTISADKSISAAYLQWSSLKASDTAMYYCARHALPNYFDSWGQGTLVTVSS ZKA218 CDRH₁ 377 GFPFSSYW CDRH₂378 INSDGRNT CDRH₃ 379 ARGGYDYDSSGCFDY VH 380EVQLVESGGGLVQPGGSLRLSCAASGFPFSSYWMHWVRQAPGKGLVWVSRINSDGRNTNYADSVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCARGGYDYDSSGCFDYWGQGTLVTV SS ZKB75 CDRH₁ 381 GFTFSNYACDRH₂ 382 ISGTGGST CDRH₃ 383 AKDSASRGGYCSGGVCYLNPGHHDY VH 384EVQVLESGGGLLQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSTISGTGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSASRGGYCSGGVCYLNPGHHD YWGQGTLVTVSS ZKB83 CDRH₁ 385GYSFTNYW CDRH₂ 386 IDPSDSYT CDRH₃ 387 ARLRGSLYCSGGRCYSVPGETPNWFDP VH 388EVQLVQSGAEVKKPGESLRISCKGSGYSFTNYWITWVRQMPGKGLEWMGSIDPSDSYTNYSPSFQGHVTISADWSINTAYLQWSSLKASDTAKYYCARLRGSLYCSGGRCYSVPGETPNW FDPWGQGTLVTVSS ZKC3 CDRH₁ 389GGSITSYY CDRH₂ 390 IYYSGST CDRH₃ 391 ARVGGAPYYYYGMDV VH 392QVQLQESGPGLVKPSETLSLTCTVSGGSITSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVGGAPYYYYGMDVWGQGTTVTVS S ZKC18 CDRH₁ 393 GFTFGDYACDRH₂ 394 IRSKAYGGTT CDRH₃ 395 SRDHTGTTYAFDI VH 396EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCSRDHTGTTYAFDIWGQGTMVTV SQ ZKD1 CDRH₁ 397 GFTFSSYGCDRH₂ 398 IWYDGSNK CDRH₃ 399 ARDRRGYGDYVGYYYGMDV VH 400QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRRGYGDYVGYYYGMDVWGQGT TVTVSS Name SEQ ID NO.Amino acid sequence ZIKV 401 TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDEDIII XQTLTPVGRLITANPVITEXTENSKMMLELDPPFGDSYIV generic IGXGXKKITHHWHRSZIKV 402 TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVD H/PF/MQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIV 2013 IGVGEKKITHHWHRS EDIIIZIKV-NS₁ 403 TGGAGTTCAACTGACGGTCG forward primer ZIKV- 404TACCCCGAACCCATGATCCT NS1- reverse primer Gapdh- 405GGCAAGTTCAAAGGCACAGTC forward primer Gapdh- 406 CACCAGCATCACCCCATTTreverse primer ZIKV 407 X₁GX₂X₃YSLCTAAFTFTKX₄PAEX₅X₆HGTVTVEX₇QY EDIIIX₈GX₉DGPCKX₁₀PX₁₁QMAVDX₁₂QTLTPVGRLITANP genericVITEX₁₃TX₁₄NSKMMLELDPPFGDSYIVIGX₁₅GX₁₆ X₁₇KITHHWHRSG whereinX₁ may be any (naturally occurring)  amino acid, preferably K, A, or E;X₂ may be any (naturally occurring)  amino acid, preferably V, F, or L;X₃ may be any (naturally occurring) amino acid, preferably S or F;X₄ may be any (naturally occurring) amino acid, preferably I or V;X₅ may be any (naturally occurring) amino acid, preferably T or V;X₆ may be any (naturally occurring) amino acid, preferably L or D;X₇ may be any (naturally occurring) amino acid, preferably V or G;X₈ may be any (naturally occurring) amino acid, preferably A or G;X₉ may be any (naturally occurring) amino acid except R, preferably Tor A; X₁₀ may be any (naturally occurring)amino acid, preferably V or I; X₁₁ may be any (naturally occurring)amino acid, preferably A or V; X₁₂ may be any (naturally occurring)amino acid, preferably M or T; X₁₃ may be any (naturally occurring)amino acid, preferably S or G; X₁₄ may be any (naturally occurring)amino acid, preferably E or K; X₁₅ may be any (naturally occurring)amino acid, preferably V or I; X₁₆ may be any (naturally occurring) amino acid, preferably E, A, K, or D; andX₁₇ may be any (naturally occurring)  amino acid, preferably E, A, or K,more preferably K or A * the sequences highlighted in bold are CDRregions (nucleotide or aa) and the underlined residues are mutatedresidues as compared to the “germline” sequence.

1-55. (canceled)
 56. Method of inhibiting a Zika virus infection in asubject, the method comprising administering to the subject an antibody,or an antigen binding fragment thereof, that specifically binds to aZika virus EDIII epitope, wherein the antibody or antigen bindingfragment thereof comprises CDRH1, CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 25, respectively. 57-109. (canceled)
 110. The method of claim56, wherein the antibody or antigen binding fragment thereof comprises aheavy chain variable region (VH) amino acid sequence having at least 70%sequence identity to the amino acid sequence of SED ID NO: 26, and alight chain variable region (VL) amino acid sequence having at least 70%sequence identity to the amino acid sequence of SEQ ID NO: 27, providedthat the antibody or antigen binding fragment thereof comprises CDRH1,CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of: (i) SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, EDN, and SEQ IDNO: 25, respectively; or (ii) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 25, respectively. 111.The method of claim 110, wherein the antibody or antigen bindingfragment thereof comprises a heavy chain variable region (VH) amino acidsequence having at least 75% sequence identity to the amino acidsequence of SED ID NO: 26, and a light chain variable region (VL) aminoacid sequence having at least 75% sequence identity to the amino acidsequence of SEQ ID NO: 27, provided that the antibody or antigen bindingfragment thereof comprises CDRH1, CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 25, respectively.
 112. The method of claim 110, wherein theantibody or antigen binding fragment thereof comprises a heavy chainvariable region (VH) amino acid sequence having at least 80% sequenceidentity to the amino acid sequence of SED ID NO: 26, and a light chainvariable region (VL) amino acid sequence having at least 80% sequenceidentity to the amino acid sequence of SEQ ID NO: 27, provided that theantibody or antigen binding fragment thereof comprises CDRH1, CRDH2,CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of: (i) SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25,respectively; or (ii) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.
 113. Themethod of claim 110, wherein the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) amino acid sequencehaving at least 85% sequence identity to the amino acid sequence of SEDID NO: 26, and a light chain variable region (VL) amino acid sequencehaving at least 85% sequence identity to the amino acid sequence of SEQID NO: 27, provided that the antibody or antigen binding fragmentthereof comprises CDRH1, CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3 aminoacid sequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ IDNO: 25, respectively.
 114. The method of claim 110, wherein the antibodyor antigen binding fragment thereof comprises a heavy chain variableregion (VH) amino acid sequence having at least 90% sequence identity tothe amino acid sequence of SED ID NO: 26, and a light chain variableregion (VL) amino acid sequence having at least 90% sequence identity tothe amino acid sequence of SEQ ID NO: 27, provided that the antibody orantigen binding fragment thereof comprises CDRH1, CRDH2, CDRH3, CDRL1,CDRL2, and CDRL3 amino acid sequences of: (i) SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25, respectively;or (ii) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 24, and SEQ ID NO: 25, respectively.
 115. The method of claim110, wherein the antibody or antigen binding fragment thereof comprisesa heavy chain variable region (VH) amino acid sequence having at least92% sequence identity to the amino acid sequence of SED ID NO: 26, and alight chain variable region (VL) amino acid sequence having at least 92%sequence identity to the amino acid sequence of SEQ ID NO: 27, providedthat the antibody or antigen binding fragment thereof comprises CDRH1,CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of: (i) SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, EDN, and SEQ IDNO: 25, respectively; or (ii) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 25, respectively. 116.The method of claim 110, wherein the antibody or antigen bindingfragment thereof comprises a heavy chain variable region (VH) amino acidsequence having at least 95% sequence identity to the amino acidsequence of SED ID NO: 26, and a light chain variable region (VL) aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 27, provided that the antibody or antigen bindingfragment thereof comprises CDRH1, CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 25, respectively.
 117. The method of claim 110, wherein theantibody or antigen binding fragment thereof comprises the heavy chainvariable region (VH) amino acid sequence of SEQ ID NO: 26 and the lightchain variable region (VL) amino acid sequence of SEQ ID NO:
 27. 118.The method of claim 56, wherein the antibody or antigen binding fragmentthereof comprises and Fc moiety.
 119. The method of claim 118, whereinthe antibody or antigen binding fragment thereof comprises a mutation inthe Fc moiety that reduces binding of the antibody or antigen bindingfragment to an Fc receptor.
 120. The method of claim 119, wherein theantibody or antigen binding fragment thereof comprises a CH2 L4Amutation, a CH2 L5A mutation, or both.
 121. The method of claim 56,wherein the antibody or antigen binding fragment thereof is comprised ina composition that further comprises a pharmaceutically acceptableexcipient, diluent, or carrier.
 122. The method of claim 56, wherein theantibody or antigen binding fragment thereof is a human antibody. 123.The method of claim 56, wherein the subject was diagnosed with Zikavirus infection or shows symptoms of Zika virus infection.
 124. Themethod of claim 56, wherein the subject is pregnant.
 125. The method ofclaim 56, wherein the antibody or antigen binding fragment thereof is anIgG1 isotype.
 126. A method of inhibiting a Zika virus infection in asubject, the method comprising administering to the subject an antibody,or an antigen binding fragment thereof, that specifically binds to aZika virus EDIII epitope, wherein the antibody or antigen bindingfragment thereof comprises CDRH1, CRDH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 25, respectively, and wherein the antibody or antigen bindingfragment thereof comprises a Fc moiety that comprises a CH2 L4Amutation, a CH2 L5A mutation, or both.
 127. A method of inhibiting aZika virus infection in a subject, the method comprising administeringto the subject an antibody, or an antigen binding fragment thereof, thatspecifically binds to a Zika virus EDIII epitope, wherein the antibodyor antigen binding fragment thereof comprises the heavy chain variableregion (VH) amino acid sequence of SEQ ID NO.: 26 and the light chainvariable region (VL) amino acid sequence of SEQ ID NO.: 27, and whereinthe antibody or antigen binding fragment thereof comprises a Fc moietythat comprises a CH2 L4A mutation, a CH2 L5A mutation, or both.
 128. Anantibody, or an antigen binding fragment thereof, that specificallybinds to a Zika virus EDIII epitope, wherein the antibody or antigenbinding fragment thereof comprises: (a) CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 amino acid sequences of: (a)(i) SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, EDN, and SEQ ID NO: 25,respectively; or (a)(ii) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, and (b)an Fc moiety comprising a mutation at at least one of E233-G236, P238,D265, N297, A327, P329, D270, Q295, A327, R292, 5239, E269, E293, Y296,V303, A327, P331, K338 and D376, and K414, wherein the mutation reducesbinding of the antibody or antigen binding fragment to an FcγRT, anFcγRIIa, an FcγRIIIa, or any combination thereof.
 129. The antibody orantigen binding fragment of claim 128, wherein the Fc moiety comprises aCH2 L4A mutation, a CH2 L5A mutation, or both.
 130. The antibody orantigen binding fragment of claim 128, wherein the Fc moiety comprises aCH2 L4A mutation and a CH2 L5A mutation.
 131. The antibody or antigenbinding fragment of claim 128, wherein the antibody further comprises amutation in the CH2 and/or CH3 domain of the heavy chain that increasesthe serum half-life of the antibody as compared to an unmutatedantibody.
 132. The antibody or antigen binding fragment of claim 128,comprising a heavy chain variable region (VH) amino acid sequence havingat least 70% sequence identity to the amino acid sequence of SEQ IDNO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 70% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 133. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 75% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 75% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 134. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 80% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 80% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 135. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 85% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 85% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 136. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 137. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 92% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 92% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 138. The antibody or antigen binding fragment of claim128, comprising a heavy chain variable region (VH) amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO.:26, and a light chain variable region (VL) amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO.:27, provided that antibody or antigen binding fragment thereofcomprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences of: (i) SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, EDN, and SEQ ID NO: 25, respectively; or (ii) SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:25, respectively.
 139. An antibody, or an antigen binding fragmentthereof, that specifically binds to a Zika virus EDIII epitope, whereinthe antibody or antigen binding fragment thereof comprises: (a) a heavychain variable region (VH) amino acid sequence SEQ ID NO: 26; (b) alight chain variable region (VL) amino acid sequence SEQ ID NO: 27; and(c) an Fc moiety comprising a mutation at at least one of E233-G236,P238, D265, N297, A327, P329, D270, Q295, A327, R292, 5239, E269, E293,Y296, V303, A327, P331, K338 and D376, and K414, wherein the mutationreduces binding of the antibody or antigen binding fragment to an FcγRT,an FcγRIIa, an FcγRIIIa, or any combination thereof.
 140. The antibodyor antigen binding fragment of claim 139, wherein the Fc moietycomprises a CH2 L4A mutation, a CH2 L5A mutation, or both.